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The  Diastatic  Enzymes  of  Wheat 

Flour  and  Their  Relation  to 

Flour  Strength 


A  THESIS  SUBMITTED  TO  THE  FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 

BY 
LOUYS  A.  RUMSEY,  B.S.,  M.  S. 

IN    PARTIAL    FULFILLMENT    OF   THE    REQUIREMENTS    FOR 
THE  DEGREE  OF  DOCTOR  OF  PHILOSOPHY 

June,  1922 


Chicago,  III. 


The  Diastatic  Enzymes  of  Wheat 

Flour  and  Their  Relation  to 

Flour  Strength 


A  THESIS  SUBMITTED  TO  THE  FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 

BY 
LOUYS  A.  RUMSEY,  B.S..,  M.  S. 

IN    PARTIAL    FULFlLLMENf    OF   THE    REQUIREMENTS    FOR 
THE  DEGREE  OF  DOCTOR  OF  THILOSOPHY 

June,  1922 


Chicago,  III. 


t'^ 


(^^^^'--i^.wHr^L- 


Research   Fellowship  Plan  Under  Which  Fellows  of  the  Americarv 

Institute  of  Baking  Have  Been  Enrolled  as  Graduate 

Students  of  the  University  of  Minnesota. 

The  American  Institute  of  Baking  in  the  fall  of  1920  detailed  two 
research  fellows  in  the  chemistry  of  baking  to  work  on  suitable  prob- 
lems as  graduate  students  of  the  University  of  Minnesota.  These 
fellows  were  regularly  registered  in  the  Graduate  School  of  the  Uni- 
versity. They  pursued  such  courses  as  ordinarily  constitute  a  study 
program  of  candidates  for,  the  doctorate  in  philosophy,  majoring  in 
the  Division  of  Agricultural  Biochesistry.  Research  problems  were 
selected  and  outlined  in  conference  with  their  advisors  in  this  Division, 
actual  work  on  the  problems  being  pursued,  by  special  agreement, 
chiefly  in  the  laboratories  of  the  American  Institute  of  Baking.  Theses 
,  based  on  this  research  were  duly  presented  in  partial  fulfillment  of 
the  degree  of  Doctor  of  Philosophy,  and  accepted  by  committees  of 
the  Graduate  School  of  the  University.  These  theses  are,  by  agree- 
ment with  the  graduate  faculty  of  the  Division  of  Agricultural  Bio- 
chemistry, published  by  their  respective  authors  as  bulletins  of  the 
American  Institute  of  Baking. 


487358 


■.; ; •';  '^ABik/OF.  ..CONTENTS 


INTRODUCTION  5 

Factors  Studied  in  Their  Relation  to  Flour  Strength 5 

Gluten  Content  5 

Protein  Analysis    5 

Colloidal    Properties   5 

Climate    ■ 6 

Electrolytes     •  6 

Electrometric  Methods    7 

Gas  Retention  and  Gas  Production 7 

Carbohydrate  Content 7 

Enzymes •  8 

Biochemical  Methods  of  Study 8 

PURPOSE  OF  INVESTIGATION 9 

HISTORICAL  10 

Definition  of  Diastase 10 

Review  of  Literature  on  Diastase 10 

Preliminary   Discussion    -17 

1.  Liquifying  and  Saccharogenic   Action 17 

2.  Resistance  of  DiflFerent  Starches  to  Diastatic  Action 19 

3.  Difference  Between  Autolytic  and  Extracted  Enzyme  Activity 20 

EXPERIMENTAL  PART  23 

1.  The  Problem 23 

2.  The  Materials    24 

3.  History  and  Description  of  Flour  Samples 25 

4.  Baking  Tests   26 

Formula  26 

Method  27 

Data  and  Score  of  Baked  Loaves 29 

5.  Clarification  of  Cereal  Extracts  and  Suspensions  by  Sodium 

Tungstate 30 

Efficiency  33 

Effect  on   Reducing  Sugar  Determination 34 

Elimination   of  Filtration    37 

Inhibition  of   Enzyme   Activity 41 

6.  Effect  of  Concentration  of  Flour-Water  Suspension  on   Diastatic 

Activity    44 

7.  Method  for  Measuring  Diastatic  Power  in  Flours 48 

8.  Effect  of  Temperature  on  the  Activity  of  Wheat  Flour  Diastase 50 

9.  Effect  of  Time  on  the  Activity  of  Wheat  Flour  Diastase 52 

10.  The  Effect  of  pH  on  the  Activity  of  Wheat  Flour  Diastase 54 

11.  Buffer  Action  and  Buffer  Valves 56 

12.  The  Relative  Diastatic  Powers  of  Fourteen  Samples  of  Wheat  Flour. 60 

13.  Diastatic   Activity   During  Fermentation  of  the   Dough 62 

Method  64 

In  Normal  Doughs 66 

In  Doughs  with  Added  Diastatic  Enzymes 66 

Discussion  of   Data 66 

THE  RELATION  OF  DIASTATIC  POWER  TO  DIFFERENT  FORMS 

OF  STARCH 70 

ANALYTICAL  DATA  ON  FLOUR  SAMPLES 73 

SUMMARY    73 

BIBLIOGRAPHY   75 

BIOGRAPHICAL   85 


THE  DIASTATIC  ENZYMES  OF  WHEAT  FLOUR 

AND  THEIR  RELATION  TO  FLOUR  STRENGTH 

By  Louys  A.  Rumsey 

INTRODUCTION 

The  role  which  the  enzymes  play  in  the  production  of  a  good  loaf 
of  bread  and  their  importance  as  contributing  factors  in  the  strength 
of  flour  is  a  matter  of  real  concern  to  both  the  baker  and  the  miller. 
After  years  of  chemical  and  physical  investigation  of  wheats  and 
flours  the  cereal  technologist  is  still  confronted  with  the  problem  of 
finding  some  factor,  or  group  of  factors,  the  determination  of  which 
will  furnish  a  sure  index  to  the  baking  strength  of  a  given  flour.  In 
spite  of  laboratory  control  in  the  blending  and  milling  of  wheats 
the  wide  variations  in  the  ability  of  the  corresponding  flour  to  produce 
a  "large,  well  piled  loaf"  (Humphries  and  Bififen,  1907.)  still  neces- 
sitates an  actual  baking  test  on  each  flour. 

Gluten  Content. — Because  of  the  unique  property  of  wheat  pro- 
teins to  form  a  tenacious,  elastic  framework  of  gluten  for  the  reten- 
tion of  the  aerating  gas,  attention  was  first  turned  to  the  amount 
and  character  of  the  gluten  content.  The  numerous  contributions 
of  Osborne  (1895  to  1907),  Guthrie  (1896),  (1900),  Fleurent  (18%), 
Snyder  (1899),  Guess  (1900),  Humphries  (1907-1910),  Guthrie  and 
Norris  (1909),  Wood  and  Hardy  (1909),  Bailey  (1916),  Blish  (1916), 
Upson  and  Calvin  (1916),  Swanson  and  Tague  (1917),  Gortner  and 
Doherty  (1918),  and  others  have  led  to  the  conclusion  that  in  the 
proteins  of  the  flour,  and  especially  in  the  gluten,  is  to  be  found  the 
most  important  basis  for  differences  in  baking  value. 

Protein  Analyses. — On  the  other  hand  Fleurent  (1901),  Snyder 
(1901),  Shutt  (1905-1908),  Norton  (1906),  Chamberlain  (1906), 
Wood  (1907),  Thatcher  (1907),  Armstrong  (1910),  Ladd  and  Bailey 
(1910),  Bailey  (1913-1914),  Blish  (1916),  and  Martin  (1920),  have 
shown  that  percentage  relation  slurps  from  protein  analyses  would  not 
suffice  for  the  predetermination  of  flour  strength.  Neither  does  the 
accurate  characterization  of  the  proteins  by  Osborne  and  his  co- 
workers apply  directly  to  the  problem  of  strength. 

Colloids. — A  more  promising  point  of  attack  appeared  to  He  in  the 
colloidal  studies  of  the  wheat  gluten,  the  work  on  which  is  well  sum- 
marized to  1918  by  Gortner  and  Doherty.  Physico-chemical  studies 
of  the  gluten  colloids  (Gortner  and  Sharp,  1921.)  involving  viscosity, 
measurements  of  the  wheat  flour  proteins  at*  different  hydrogen  ion 
concentrations,  being  conducted  in  the  Division  of  Agricultural  Bio- 


chemistry,  I^Srtivemt)!  of  Min]ri^ota,.give  promise  of  furnishing  a  most 
valuable  index  of  strength  for  those  flours  in  which  strength  is  deter- 
mined by  gluten  quality. 

Climate. — The  experience  of  the  millers  as  well  as  the  direct  ex- 
perimental evidence  summarized  by  the  reports  of  LeClerc  (1909, 
1910,  1912,  1914),  Bailey  (1913),  and  Thatcher  (1913),  have  demon- 
strated that  environment  is  the  most  important  factor  in  the  develop- 
ment of  the  wheat  kernel.  The  biological  differences  in  the  rela- 
tionships of  various  constituents  are  most  marked  when  the  same 
seed  is  grown  under  different  climatic  conditions.  While  changes  of 
soil  impose  certain  changes  in  the  protein,  ash,  and  carbohydrate  con- 
tent of  the  same  wheats,  the  correlation  is  by  no  means  as  simple 
and  direct  as  that  between  climate  and  "strength"  of  the  wheat  flour. 
Likewise  different  varieties  and  types  of  wheat  respond  most  readily 
to  climatic  changes.  The  corresponding  "strength"  characteristics 
have  consequently  come  to  be  regarded  as  typical  of  certain  grow- 
ing regions.  We  should  expect  the  same  changes  to  be  manifested 
in  the  respective  enzymes  of  the  wheat  kernel  as  carried  over  into 
the  flour.  For  instance,  a  hard  spring  wheat,  cut  and  shocked  be- 
fore it  is  biologically  ripe,  should  be  expected  to  show  a  different 
diastase  activity  than  a  softer  wheat  grown  in  the  dry  and  semi-arid 
regions  where  the  "dead  ripe"  grain  is  allowed  to  stand  in  the  field 
under  a  hot  sun  and  hot  winds  for  days  before  cutting  and  thresh- 
ing. Humphries  (1910)  came  to  the  conclusion  that  such  climatic 
factors  did  operate  so  as  to  change  the  diastatic  activity  of  the  flours 
from  these  wheats  as  well  as  their  other  strength   characteristics. 

Electrolytes. — The  addition  of  various  electrolytes  as  flour  "im- 
provers" and  as  yeast  nutrients,  bears  a  close  relationship  to  the  prob- 
lem of  gluten  quality.  As  early  as  1905  Fleurent  began  to  inves- 
tigate the  effects  of  different  acids  and  salts  on  gluten.  Wood  (1907), 
Shutt  (1908),  Wood  and  Hardy  (1909),  Humphries  (1910),  Wil- 
lard  and  Swanson  (1912),  and  Olson  (1917),  are  among  the  earlier 
contributors  to  the  study  of  electrolytes  and  their  relation  to  strength. 
Numerous  experiments  with  flour  "improvers"  and  yeast  stimulants 
have  furnished  a  basis  for  the  widespread  use  of  various  electrolytes 
on  a  commercial  scale  for  the  improvement  of  the  baked  loaf.  The 
most  of  these  investigations  are  included  in  the  reports  of  Jessen- 
Hansen  (1911),  Bacon  (1916),  Kohman  (1915),  and  (1916)',  Koh- 
nian,  Hoffman,  et  al  (1916),  Hoffman  (1917),  Thomas  (1917),  Hen- 
derson, Fenn,  and  Cohn  (1919),  and  Henderson,  Cohn,  Cathcart, 
Wachman  and  Fenn  (1919).  The  real  action  of  such  added  ma- 
terials, for  example  on  the  gluten  or  on  the  enzymes,  is  but  poorly 
understood   as   yet,   and   the   whole   subject   needs   further   research. 


Electrometric  Methods. — The  introduction  of  electrometric  meth- 
ods into  the  field  of  biological  research  has  both  facilitated  and  stim- 
ulated new  methods  of  attack  on  the  problem  of  flour  strength.  De- 
terminations of  the  hydrogen  ion  concentration  of  flour,  dough,  and 
bread  have  helped  to  clear  up  some  vexing  questions,  the  most  notable 
of  which  are:  the  control  of  undesirable  organisms  in  bread,  Cohn 
and  Henderson  (1918),  Cohn,  et  al  (1918),  Morrison  and  Collatz 
(1921);  the  optimum  acidity  for  proper  fermentation,  Jessen-Han- 
sen  (1911),  Cohn,  Cathcart  and  Henderson  (1918);  the  optimum 
hydrogen  ion  concentration  for  enzyme  actions,  Sherman  and  Wal- 
ker (1917),  Sherman,  Thomas  and  Baldwin  (1919)  ;  and  the  lack  of 
relationship  between  true  and  titrable  acidity,  Swanson  and  Tague 
(1919).  Quite  recently  Bailey  (1918),  Bailey  and  Collatz  (1921), 
and  Bailey  and  Peterson  (1921),  have  applied  electrometric  meas- 
urements successfully  to  the  determination  of  electrolyte  content 
of  flours  in  their  relation  to  flour  grade.  Collatz  and  Bailey  (1921), 
have  likewise  been  able  to  measure  results  of  phytase  activity 
by  means  of  electrical  conductivity  measurements. 

Gas  Retention  and  Gas  Production. — Wood  in  1907,  Humphries 
in  his  discussion  of  Baker  and  Hulton's  paper  on  flour  strength 
1908,  and  Armstrong  in  1910,  all  emphasized  their  opinions  as 
to  the  importance  of  gas  retention  by  a  dough  in  relation  to  the  gas 
production  during  fermentation.  In  1916  Bailey  proposed  a  method 
for  comparative  measurements  of  this  ratio  by  means  of  an  "ex- 
pansimeter".  Martin  (1920)  has  published  the  results  of  compara- 
tive tests  on  the  gas  producing  capacity,  and  gas  retaining  power 
of  flours  based  on  the  fermentation  method  of  Wood.  He  summarizes 
these  two  factors  in  flour  strength  by  ascribing  to  a  strong  flour 
a  minimum  gas-producing  capacity  with  a  high  gas-retaining  capac- 
ity. Bailey  and  Weigley  (1922)  have  just  completed  a  report  of  some 
new  studies  on  the  retention  of  carbon  dioxide  in  doughs  in  relation 
to  gas  production  which  lead  to  the  interesting  preposition  that  part 
of  the  ripening  of  the  dough,  and  much  of  the  spring  in  the  oven 
is  due  to  the  carbon  dioxide  gas  dissolved  in  the  dough.  Also  their 
conclusions  relative  to  the  ratfo  of  gas  production  to  gas  retention 
factors  are  in  agreement  with  the  hypothesis  of  Humphries,  Arm- 
strong, Biffen  and  others  that  the  carbon  dioxide  production-rate 
is  one  of  the  important  factors  in  flour  strength. 

Carbohydrate  Content  of  Flour. — The  intimate  relationship  be- 
tween alcoholic  fermentation  and  the  proper  aeration  of  the  dough 
necessitated  data  on  the  carbohydrate  content  of  wheat  flour.  But 
the  analyses  of  Teller  (1898),  (1912),  Stone  (1896  and  1897),  Brown, 
Morris,  and  Millar  (1897),  Konig  (1898),  Shutt  (1907,  1908),  Alway 


and  Hartzell  (1909),  Liebig  (1909),  Jago  (1911),  Thatcher  (1913, 
1915),  Olson  (1917),  all  verify  the  same  conclusion,  namely,  that 
the  small  amounts  of  soluble  carbohydrates  naturally  occurring  in 
flour  are  insignificant  in  comparison  to  the  amounts  required  for 
proper  fermentation  in  the  dough  batch. 

It  should  be  borne  in  mind  that  there  may  be  some  correlation  be- 
tween the  natural  sugar  content  of  the  wheat  and  its  relative  state 
of  biological  ripeness.  The  investigations  of  Teller,  Shutt,  Thatcher, 
and  Olsen,  just  referred  to,  would  indicate  the  efifect  of  climatic  factors 
on  the  sugar  content  in  flour  milled  from  certain  wheats. 

Enzymes. — A  natural  corollary  to  the  study  of  carbohydrate  content 
is  the  investigation  of  those  enzymes  which  are  responsible  for  the  pro- 
duction of  carbohydrates  available  for  yeast  growth  and  fermenta- 
tion, and  those  which  alter  and  "condition"  the  gluten.  Wood  (1907), 
and  Humphries  (1907,  1910),  Liebig  (1909),  recognized  the  greater 
importance  of  diastatic  action  than  of  sugar  content  in  panary  fer- 
mentation, and  they  reported  measurements  of  the  activity  of  such 
enzymes.  These  earlier  experiments,  along  with  other  preliminary 
work  on  the  enzymes  of  wheat  flour  bore  evidence  of  their  impor- 
tance as  contributory  factors  in  the  production  of  a  good  loaf.  In  addi- 
tion to  diastase,  the  other  enzymes  which  have  received  the  most 
study  relative  to  their  action  in  flour  and  dough  are  the  proteases, 
the  phytase,  and  catalase.  The  presence  of  a  cytase,  acting  upon 
the  outer  surface  or  envelope  of  starch  cellulose,  has  been  postulated, 
but  the  evidence  for  the  activity  of  this  enzyme  in  flour  is  incon- 
clusive. For  the  purpose  of  this  report  the  activities  of  these  dif- 
ferent enzymes  can  best  be  considered  along  with  the  discussion 
of  diastase. 

Strength  Probably  Due  to  the  Interaction  of  Several  Factors. — It 
has  become  evident  from  the  accumulated  data  resulting  from 
years  of  investigation  that  no  single  analytical  factor  has  yet  been 
found  which  will  suffice  to  predetermine  the  baking  strength  of  a 
given  flour.  The  baking  test  cannot  as  yet  be  replaced  by  any  accurate 
means  of  predetermining  baking  value.  It  therefore  becomes  neces- 
sary to  continue  the  studies  of  the  various  factors  which  go  to  make 
up  this  property  of  flour  strength,  not  by  themselves,  but  relative- 
ly and  collectively. 

Biochemical  Method. — The  wheat  berry  containing  the  plant  em- 
bryo and  associated  food  stored  there  for  nourishment  of  the  plantlet 
is  produced  by  the  plant  for  the  propagation  of  its  own  species.  The 
commercial  flours  produced  by  the  roller  milling  process,  and  known 
as  patent,  straight,  first  and  second  clears,  contain  about  70  to  80  per- 
cent of  the  wheat  berry,  mostly  of  the  food  storage  material,  with 

8 


increasing  amounts  of  the  branny  coverings  and  fragments  of  em- 
bryo, in  the  order  named.  Any  differences  in  the  laying  down  or 
''setting"  of  this  storage  material,  or  in  the  development  of  the  em- 
bryonic elements,  were  imposed  by  the  biological  changes  during 
growth,  ripening,  and  storing  of  the  wheat,  and  will,  therefore,  be 
reflected  in  the  quality  of  the  flour  after  milling.  Studies  on  the 
progressive  development  of  the  wheat  kernel  by  Teller  (1898),  Wym- 
per  (1909),  Brenchley  and  Hall  (1909),  Thatcher  (1913,  1915),  Eck- 
erson  (1917)  ;  and  those  on  the  chemical  changes  in  wheat  under 
various  conditions  of  storage  or  handling  by  Swanson  (1916),  Olson 
(1917),  Bailey  and  Gujar  (1918-1920),  and  Blish  (1920),  will  justify 
our  belief  in  the  dependence  of  flour  quality  upon  biological  bases. 
The  problem  of  flour  strength  must,  therefore,  be  approached  from 
the  bio-chemical  point  of  view:  first,  to  gather  all  the  available  in- 
formation concerning  the  character  and  properties  of  each  individ- 
ual biological  factor  contributing  to  flour  strength;  and  second,  to 
study  these  factors  relatively  and  collectively  in  their  many  and  labile 
relationships.  The  factors  to  be  controlled  in  such  an  undertaking 
appear  to  be  almost  without  limit,  and,  therefore,  the  task  of  a  collective 
study  of  flour  strength  to  be  almost  hopeless.  Yet  by  judicious  se- 
lection of  constants  and  variables,  a  beginning  is  made  which  au- 
tomatically leads  to  a  better  knowledge  of  the  relationships  of  pro- 
tein content,  gluten  quality  as  shown  by  viscosity  and  other  colloidal 
properties,  enzyme  activity,  carbohydrate  content,  gas  retaining  abil- 
ity, yeast  activity  and  fermetation,  effect  of  shortening  agents,  acid- 
ity, buffer  action  of  salts,  chemical  "improvers"  and  yeast  stimulants, 
temperature,  time,  etc. 

Purpose'. — While  this  investigation  was  undertaken  primarily  to 
set  aside  and  study  in  detail  the  diastatic  enzymes  of  flours  in  their 
relation  to  flour  strength,  the  plan  also  included  the  bringing  to- 
gether of  as  many  controlled  factors  as  possible  to  bear  on  a  series 
of  selected  flours.  The  data  published  by  earlier  workers  had  been 
largely  confined  to  the  study  of  one  or  two  factors  on  a  limited  num- 
ber of  flours,  largely  because  of  the  enormous  expenditure  of  time  and 
energy  required  to  make  a  more  collective  study.  These  flours  can- 
not later  be  duplicated.  And  inasmuch  as  flours  differ  so  widely 
among  themselves  it  has  been  difficult  for  another  worker  to  take 
up  the  problem  and  secure  comparative  results  even  when  the  same 
technique  is  employed.  This  applies  even  more  strongly  when  at- 
tempts are  made  to  correlate  data  by  different  workers  on  different 
factors.  It  was  hoped  to  overcome  this  difficulty  to  some  extent  in 
the  present  work,  first,  by  collecting  fifteen  samples  of  flour  milled 
from  authentic   samples   of  wheat  and  representing   various   typical 


wheat  producing  areas  of  North  America;  second,  by  the  cooper- 
ative work  of  three  investigators  working  on  the  same  set  of  samples 
but  from  different  points  of  attack;  and  third,  by  a  complete  record 
of  all  the  available  analytical  data  obtained  on  each  flour.  The  co- 
operation of  the  American  Institute  of  Baking  research  laboratories 
with  the  Division  of  Agricultural  Biochemistry  of  the  University  of 
Minnesota  made  such  a  plan  possible.  The  work  on  this  series  of  flours 
has  now  extended  over  nearly  two  years.  The  present  paper  is  the  first 
report.  Additional  factors  wlill  be  considered  by  the  other  col- 
laborators in  subsequent  publications. 

HISTORICAL 

Payen  and  Perzos  in  1833  proposed  the  term  diastase  to  designate 
that  active  agent  in  malt  which  transformed  starch  to  dextrins  and 
some  form  of  sugar.  Jago  (1911)  further  characterized  as  diastase 
that  agent  of  malt  which  transforms  starch  or  soluble  starch  to  mal- 
tose. In  the  voluminous  literature  on  the  subject  of  amylase  and  dias- 
tase, the  terms  are  sometimes  used  interchangeably.  Later  tendency 
is  to  distinguish  between  amyloclastic  and  saccharogenic  action  de- 
pending upon  which  activity  is  to  be  measured.  Unfortunately  this 
differentiation  has  not  been  as  general  as  might  be  desired. 

Definition  of  Diastase. — Leaving  a  consideration  of  the  character 
of  the  two  enzymes  involved  for  later  discussion,  it  should  be  stated 
here  that  the  term  diastase  is  restricted  throughout  this  paper  to 
mean  "That  enzyme  or  group  of  enzymes  which  through  their  suc- 
cessive or  cumulative  action  produce  maltose  from  the  starch  avail- 
able." 

It  is  impractical  to  consider  in  detail  the  great  mass  of  literature 
on  the  subject  of  diastase  in  the  malting  and  brewing  industries  ex- 
cept as  it  applies  directly  to  the  problem  of  better  bread. 

In  1907  T.  B.  Wood  published  the  results  of  some  studies  on  the 
the  factors  influencing  the  size  and  shape  of  the  loaf.  His  paper 
marks  the  transition  from  the  purely  chemical  analytic  to  a  biological 
point  of  view.  Although  a  sharp  distinction  was  not  drawn  between 
the  amount  of  sugars  in  the  flour  and  those  produced  by  diastatic  ac- 
tion, he  clearly  recognized  "that  the  sugar  so  formed,  together  with  that 
originally  present,  forms  the  source  from  which  the  yeast  makes  the 
carbon  dioxide  it  produces  when  the  dough  is  fermented."  This  re- 
fers to  the  baking  process  then  prevalent  in  England  in  which  little 
or  no  added  sugar  was  used  in  the  dough  batch,  the  fermentation 
depending  almost  entirely  upon  the  sugars  produced  by  diastatic 
action.  To  arrive  at  some  measure  of  the  sugars  thus  available  for 
yeast  fermentation,  20.  grams  of  flour  with  20  cc  of  water  and  0.5 

10 


grams  of  yeast  were  incubated  at  35°C,  and  the  volume  of  carbon 
dioxide  given  off  was  measured  over  a  period  of  hours.  The  result- 
ing data  led  to  the  conclusion  that:  "Here  too  the  rate  of  gas  evolu- 
tion and  the  size  of  the  loaf  run  parallel,  and  it  seems  certain,  there- 
fore, that  it  is  more  particularly  the  gas  given  off  in  the  later  stages 
of  dough  fermentation  that  determines  the  size  of  the  loaf.  This 
being  so  the  size  of  the  loaf  will  depend,  not  so  much  on  the  sugar 
present  in  the  flour  as  such,  as  on  the  diastatic  capacity,  which  will 
cause  continued  sugar  formation,  and  consequently  continued  gas 
evolution  in  the  dough.  Probably,  therefore,  measurement  of  the  gas 
evolved  in  the  later  stages  of  the  fermentation  would  give  a  more 
accurate  test  for  the  power  of  making  a  large  loaf  than  the  measure- 
ment which  I  have  made  of  the  total  volume  given  off  in  24  hours." 
Shutt  (1907)  in  commenting  on  Wood's  paper,  expressed  a  belief, 
founded  on  analysis  of  sugars  in  flours,  that  diastatic  activity  rather 
than  the  sugar  content  in  a  flour  is  the  determinative  factor  in  loaf 
volume.  His  limit  for  reducing  sugar  is  .62%  and  for  non-reducing 
sugar  1.22%.  A  few  months  later  there  appeared  the  contributions 
of  Baker  and  Hulton  (1908)  and  Ford  and  Guthrie  (1908).  These 
two  investigations,  made  independently  of  each  other,  were  the  first 
attempts  at  a  systematic  study  of  the  enzymes  of  wheat  flour  in 
their  relation  to  baking  value.  Ford  (1904)  had  previously  considered 
the  estimation  of  diastatic  power  in  malt  flours,  using  soluble  starch 
as  a  substrate.  Many  necessary  precautions  were  pointed  out  con- 
cerning the  preparation  of  pure  soluble  starch,  and  the  effect  of 
temperature,  time,  and  acidity  on  the  extraction  of  diastase,  so  that 
the  results  of  this  oft  quoted  article  have  an  important  bearing  on 
the  determination  of   diastatic  activity  in  wheat  flour. 

Baker  and  Hulton's  statement  of  the  importance  of  diastase  action 
in  fermentation  is  in  agreement  with  that  of  Wood  as  quoted  above.  . 
They  said  "It  is  certain  that  some  of  the  carbon  dioxide  concerned 
in  the  rise  of  bread,  especially  in  the  later  stages  of  doughing  and 
in  the  earlier  period  of  baking,  is  formed  from  the  fermentation  of 
the  maltose  produced  by  the  action  of  the  diastase  on  the  flour 
starch."  They  demonstrated  the  presence  of  diastatic  action  in  a 
dough  by  extracting  the  sugars  and  preparing  the  osazones  of 
glucose,  and  glucose  and  maltose,  respectively,  from  two  doughs 
made  without  yeast,  one  of  which  had  the  diastase  inactivated  by 
.02N  NaOH,»the  other  allowed  to  remain  active  for  four  hours  at 
40°C.  But  their  measurements  of  the  activity  of  flour  extracts  on 
soluble  potato  starch,  although  indicating  its  similarity  to  barley 
diastase  (Baker,  J.  L.,  1909),  showed  that  large  differences  exist 
between  this  activity  of  extracts  and  the  activity  of  the  enzyme-con- 

11 


taining  tissues  when  in  the  state  of  a  dough,  and  it  was  recognized 
that  this  work  on  soluble  starch,  after  the  method  of  Lintner,  gave 
no  measure  of  liquifying  enzymes.  They  found  by  measuring  the 
amount  of  carbon  dioxide  produced  during  fermentation  of  a  dough, 
as  suggested  by  Wood,  that  there  was  a  close  correlation  of  carbon 
dioxide  production  to  maltose  resulting  from  diastatic  activity,  cor- 
recting for  original  sugars  present.  They  also  pointed  out,  however, 
that  "a.  weak  flour  may  have  a  diastatic  power  as  high  as  or  even 
higher  than  a  strong  flour." 

The  conclusions  drawn  from  data  on  carbon  dioxide  production 
are  of  especial  interest  because  with  the  exception  of  the  few  trials 
by  Olson  (1917),  no  results  have  been  obtained  for  the  liquifying 
agent  in  diastase  of  wheat  flour.  To  quote  from  his  conclusions : 
"The  results  indicate,  we  think,  most  conclusively  that  the  low  gas 
production  of  this  flour  (a  weak  flour)  arises  fron  an  inadequate 
supply  of  starch  liquifying  enzyme.  We  have  already  shown  that 
the  gas  to  diastase  ratio  is  higher  on  the  whole  in  strong  flours,  and 
it  seemed  probable,  in  view  of  the  last  experiment,  that  we  might 
establish  a  connection  between  the  strength  and  the  relative  amount 
of  a  starch  liquifying  enzyme  in  a  flour." 

The  same  phase  of  this  problem  was  approached  from  a  dififerent 
angle  by  Ford  and  Guthrie  (1908).  Most  of  their  efforts  were  directed 
toward  obtaining  complete  extraction  of  the  diastase  from  flour  by 
applying  the  methods  previously  used  for  barley  diastase.  The  effect 
of  both  added  active  proteoclastic  enzymes,  such  as  "papaien,"  and 
of  various  concentrations  of  added  salts,  such  as  sodium  chloride, 
potassium  chloride,  and  phosphates,  were  tried,  resulting  in 
the  increase  of  diastatic  activity  in  the  extract.  They  state:  "The 
amylase  of  wheat,  like  highly  purified  preparations  of  amylolytic 
enzymes  from  other  sources  does  not  exhibit  its  full  hydrolytic 
activity  except  in  presence  of  a  certain  salt  concentration."  This 
same  principle  of  salt  effect  in  the  protection  of  diastatic  action 
was  made  use  of  by  Sherman  and  Walker  (1917)  and  Sherman, 
Thomas  and  Baldwin  (1919)  in  their  studies  on  the  amylases  of  ani- 
mal, plant  and  fungus  origin. 

The  data  of  Ford  and  Guthrie  for  the  diastatic  activity  of  their  po- 
tassium chloride  extracts  from  twelve  different  flours  showed  but 
slight  differences  in  value  as  maltose  per  gram  of  flour.  They  carried 
autolytic  measurements  further  than  Baker  and  Hulton  had  done, 
but  with  the  object  of  determining  what  effect  the  proteoclastic  ac- 
tion would  have  on  the  diastase.  After  allowing  the  1  :  25  flour  water 
suspension  to  digest  autolytically  for  different  periods  of  time  at  30°C. 
the  diastase  was  extracted  and  its  action  on  2%  soluble  starch  was 

12 


After     3  hours 

2.87 

After    4  hours 

2.80 

After     5  hours 

2.66 

After  26  hours 

2.52 

measured.  The  resulting  data  which  are  presented  here  as  Table 
1  shows  the  peculiar  fact  that  such  a  procedure  gives  practically 
a  constant  diastatic  activity  regardless  of  time.  The  results  of  That- 
cher and  Koch  which  are  discussed  later,  showed  similar  results 
with  the  same  sort  of  treatment. 

TABLE  I. 
"Autodigestion  of  Wheaten  Flour" 
•(The  digestion  of  soluble  starch  by  diastase  extracted  from  flour- 
water  suspensions  which  had  been  allowed  to  digest  autolytically  for 
different  periods  of  time):  (Ford  &  Guthrie,  1908). 

Grams  Maltose  per  1  g.  Flour 
Flour  No.  1  Flour  No.  2 

After     1  hour  2.87  11.83 

11.90 
11.69 
11.34 
12.74 

One  noteworthy  suggestion  in  this  contribution  is  that  perhaps  the 
degradation  of  the  gluten  in  weaker  flours  by  the  proteases  in  some 
diastatic  malt  preparations  is  the  reason  for  their  failure  to  improve 
the  quality  of  the  loaf.  Confirmation  of  this  hypothesis  still  awaits 
further  research  on  the  proteases  of  wheat  and  malt  flours.  Pre- 
liminary investigations  along  this  line  in  this  laboratory  point  to  the 
undoubted  importance  of  proteases  as  factors  in  panary  fermentation. 

Baker  and  Hulton  also  had  considered  the  proteoclastic  enzymes 
of  the  flour,  but  expressed  the  opinion  that  these  enzymes  do  not  ex- 
ert sufficient  effect  on  the  gluten  during  the  baking  period  to  be  of 
serious  consequence  in  the  baking  value  of  a  flour.  On  the  other 
hand,  how^ever,  they  point  out  that  the  proteoclastic  enzyme  of  the 
yeast  probably  play  a  more  important  part  in  the  modification  of 
the  gluten  during  fermentation.  This  fact  was  demonstrated  by  the 
presence  of  2.7%  of  soluble  nitrogen  (as  protein)  in  a  fermented 
dough  as  compared  to  1.9%  in  the  same  dough  made  without 
yeast.  Liebig  in  1909  reported  the  results  of  comparative  sugar  de- 
terminations made  on  wheat  meal  and  on  doughs  made  of  the  same 
meal.  He  found  that  maltose  w^s  steadily  produced  by  the  action  of 
diastatic  enzymes  in  a  dough  after  standing  fourteen  hours  at  30  to 
40  degrees,  and  the  reducing  sugar  content  calculated  as  dextrose 
amounted  to  4.6  percent.  Doughs  fermented  two  hours  with  yeast 
showed  an  excess  of  unfermented  reducing  sugar  which  Liebig  con- 
cluded was  due  to  the  action  of  diastase. 

There  w^as  a  great  deal  of  general  interest  shown  by  the  chemists 
at  that  time  in  the  diastatic  and  proteoclastic  enzymes  of  wheat 
flour.  Neumann  and  Salecker  (1908),  and  Kohman  (1909),  demon- 
strated  the  improvement  of  bread  by   the  addition   of  active   dias- 

13 


tatic  malt  preparations.  Humphries  (1910)  stated  very  clearly  the 
position  of  diastase  as  a  factor  in  flour  strength.  Armstrong  (1910) 
likewise  discussed  its  practical  importance  in  the  bakery  but  he 
doubted  the  possibility  of  correlating  diastatic  power  with  flour 
strength.  Armstrong  objected  to  Wood's  (1907)  measurements  of 
gas  production  and  diastatic  power  because  "they  were  made  under 
conditions  very  different  from  those  which  prevail  in  actual  bakehouse 
practice." 

Most  of  the  earlier  data  in  the  literature,  while  significant,  is  of 
less  value  than  its  accompanying  discussion.  This  is  true  chiefly 
because  of  the  difficulty  in  controlling  the  conditions  necessary  for  a 
study  of  the  wheat  flour  enzymes. 

Sherman,  Kendall  and  Clark  (1910),  reviewed  the  various  methods 
for  use  in  the  determination  of  diastatic  power  as  a  preliminary  to  a 
careful  series  of  studies  on  the  amylases  of  different  origin.  The 
method  and  the  scale  adopted  by  them  depended  upon  the  weight  of 
maltose  produced  by  the  action  of  the  diastatic  preparation  on  soluble 
starch  at  40°C  for  30  minutes.  Sherman's  prediction  (1910)  that 
the  determination  of  diastatic  power  would  "soon  become  an  impor- 
tant factor  in  the  valuation  of  commercial  American  Malts."  has 
become  a  fact.  The  registration  of  diastatic  malt  preparations  for 
sale  to  the  baking  trade  has  shown  the  necessity  for  practical,  uni- 
form, standard  methods  of  measurement.  (American  Institute  of 
Baking,  1921.)  The  measurement  of  diastatic  power  as  carried  out 
at  the  present  time  among  the  control  laboratories  of  this  country 
show  the  most  surprising  variations  in  method  and  result.  The  Lint- 
ner  method  (1886),  or  one  of  its  modifications,  is  still  the  basis  for 
most  of  them. 

The  most  important  data  in  the  literature  to  date  concerning  the 
autolytic  diastatic  activity  in  wheat  flours  is  that  of  Swanson  and 
Calvin  (1913).  They  stated:  *Tf  flour  itself  possesses  such  large  di- 
astase activity  that  by  digesting  it  with  water  at  a  suitable  tempera- 
ture, more  than  one  fifth  of  its  weight  is  transformed  into  soluble 
carbohydrates  such  as  glucose  and  maltose,  the  conditions  for  such 
transformations  deserve  careful  study."  These  authors  allowed  the 
diastase  of  the  wheat  flour  to  act  autolytically  on  the  wheat  starch 
in  water  suspensions  of  various  concentrations,  measuring  the  amount 
of  maltose  produced.  This  procedure  v^ould  appear  to  give  an  index 
of  the  activity  to  be  expected  in  a  dough.  But  no  use  has  been 
made  of  this  valuable  data  in  the  improvement  of  baking  science. 
It  is  now  impossible  to  duplicate  the  flours  they  used;  nevertheless, 
the  data  can  be  considered  as  quite  comparable  to  that  obtained  on 
other  flours  of  similar  quality,  and   so  may  be  used  on  that  basis. 

14 


The  problem  of  diastase  in  the  cereal  grains  was  again  attacked  by 
Thatcher  and  Koch  in  1914.  Instead  of  applying  auto-digestion  as 
did  Swanson  and  Calvin,  they  undertook  to  obtain  extracts  of  constant 
diastatic  power  by  digestion  of  the  ground  cereal  with  water  at 
0°C  for  two  hours.  This  extract  was  then  filtered  in  the  cold, 
allowed  to  act  on  soluble  starch  for  30  minutes  at  40° C,  and  the 
resulting  maltose  determined.  The  tables  of  results  show  some 
variation  with  time  of  extraction,  which  was  due  to  the  loss  of  dias- 
tatic power  with  a  corresponding  increase  of  sugars  even  at  the  low 
temperatures  employed.  Comparative  results  for  one  to  five  hour 
extractions  could  only  be  obtained  because  the  time  curve  for  dias- 
tatic action  at  0°C  rises  so  slowly  that  differences  between  one  and 
three  hours  were  within  the  experimental  error  of  the  methods  em- 
ployed (Fig.  2). 

The  probable  error  of  a  determination  based  on  30  minutes  action 
at  40° C  is  large,  because  this  point  occurs  on  the  steeper  part  of  the 
curve,  where  differences  of  1°C  or  1.  minute  in  time  is  considerable. 
The  fact  that  no  significant  differences  were  observed  betw'een 
medium  and  fine  grinding  of  the  materials  apparently  eliminates  at 
least  one  possible  variant  in  the  study  of  cereal  diastase.  Their 
results  confirm  the  precautions  necessary  in  the  determination  of  malt 
diastase  as  pointed  out  by  Kjeldahl,  Ford,  Brown  and  Glendening,  and 
others,  namely,  there  must  be  present  so  large  an  excess  of  starch 
that  less  than  40%  is  hydrolyzed  to  sugars. 

Swanson,  with  Fitz  and  Dunton,  published  in  1916  the  results  of 
milling  and  baking  tests  on  wheats  which  had  been  subjected  to 
different  methods  of  handling  and  storing.  The  effect  of  increased 
diastatic  enzymes  by  the  addition  of  small  iamounts  of  germinated 
wheat  flour  was  to  increase  the  "spring"  in  the  oven  and  increase  the 
volume  of  the  loaf.  Too  large  amounts,  or  flour  from  wheat  which 
had  germinated  over  too  long  a  period,  resulted  in  a  weakened 
gluten,  poor  texture,  and  an  inferior  loaf  of  bread.  More  conclusive 
data  was  published  the  next  year  by  Olson  (1917),  who  made  com- 
parative baking  tests  on  strongly^ diastatic  flours  milled  from  germin- 
ated wheat.  No  measurements  were  made  on  the  relative  saccharo- 
genic  powers  of  these  flours,  but  their  amyloclastic  powers  were  com- 
pared by  incubating  the  1  :  20  flour-water  mixture  at  70° C.  until  the 
iodine  test  failed  to  show  the  characteristic  blue  color.  The  increase 
of  amylase  activity  in  the  flour-  from  germinated  grain  with  a  one 
centimeter  epicotyl  as  compared  to  that  from  grain  which  had  only 
started  to  germinate,  was  remarkable.  Loaves  baked  from  the  germ- 
inated wheat  flour  showed  increases  in  volume,  but  those  containing 
too  much  amylase  suffered  in  texture  and  character  of  crumb  because 

15 


of  too  great  liquifying  action.  This  dextrinizing  action  supports 
Humphries'  (1910)  contention  that  added  diastase  in  small  quantity 
had  the  property  of  very  perceptibly  improving  the  flavor  of  a  loaf, 
probably  due  to  the  correlation  of  increased  dextrin  content  and  the 
consequently  improved  conditions  for  the  retention  of  moisture  within 
the  loaf.  Olson  showed  further  that  the  addition  of  varying  small 
quantities  of  these  diastatic  flours  to  a  normal  flour  did  increase  the 
Welter-holding  capacity  in  direct  proportion  to  the  amount  of  germin- 
ated wheat  flour  added.  In  one  case  the  volume  of  the  loaves  were 
increased  as  much  as  49%  without  apparent  impairment  of  the  quality 
of  bread,  and  in  all  cases  there  was  an  increase  in  volume,  but  the 
important  fact  in  this  connection  depends  upon  the  inherent  "strength" 
of  the  normal  flour  used.  The  gluten  of  some  weaker  flours  is  of 
such  poor  quality  that  it  canno.t  retain  the  extra  gas  produced  through 
the  agency  of  an  added  excess  of  diastase,  and  consequently  the  tex- 
ture of  the  loaf  suffers.  It  appears  from  those  results  that  added 
diastase  within  certain  limits,  improves  the  baking  quality  of  a  flour, 
but  the  inherent  strength  of  the  flour  governs  the  quantity  of  diastase 
which  may  be  used.  These  conclusions  agree  with  the  work  of 
Humphries,  Kohman,  Neumann  and  Salecker. 

The  data  on  the  variations  of  the  different  forms  of  nitrogen  com- 
pounds in  the  germinated  wheat  flours  also  lead  one  to  suspect  that 
perhaps  the  proteoclastic  enzymes,  in  their  degradation  of  the  gluten, 
have  had  a  more  profound  influence  in  the  impairment  of  the  grain 
and  texture  of  the  poorer  loaves  than  the  action  of  the  diastase.  Con- 
firmation of  this  probability  must  await  further  research  on  the  ac- 
tivity of  proteoclastic  enzymes  and  their  effect  in  baking.  The  recent 
work  of  F.  J.  Martin  (1920)  is  in  effect  a  continuation  of  the  investiga- 
tions of  Wood  (1907).  The  volumes  of  gas  given  off  by  fermentation 
of  20.  grams  of  flour  with  55%  water,  1.2%  salt,  and  1.0%  yeast,  over 
a  period  of  24  hours  at  29° C  were  measured  at  different  points  of  time. 
The  volumes  of  the  dough  were  in  close  relation  to  the  gas  production 
during  the  first  periods  of  fermentation.  But  after  a  normal  fermen- 
tation period  of  about  three  hours  the  relation  was  complicated  by 
the  reduction  of  the  gas  retaining  power  of  the  dough,  due  to  a 
weakening  of  the  gluten.  There  are  several  points  in  Martin's  paper 
which  are  of  significance  in  a  study  of  enzymatic  activities  in  flour. 
The  variation  in  the  volumes  of  gas  produced  by  the  different  flours 
at  any  stage  of  fermentation  was  not  great,  and  could  not  be  cor- 
related closely  with  final  loaf  volume.  But  in  the  case  of  a  weak 
flour,  one  which  showed  a  low  gas-producing  power,  the  deficiency 
of  gas  production  in  the  later  stages  of  fermentation  could  be  rectified 
by  the  addition  of  diastatic  enzymes,  with  a  consequent  increase  in 

16 


volume  of  the  loaf.  These  results  are  in  agreement  with  those  of 
Kohman  and  Olson.  The*  analytical  data  on  Martin's  flours  offer 
some  interesting  comparisons.  They  would  appear  to  show  the  pos- 
sibility of  obtaining  a  constant  for  the  ratios  between  water  soluble 
proteins,  gas  retaining  powers,  and  bakers'  marks.  The  difference 
between  his  gliadin  figures  and  the  so-called  "amended  gliadin"  point 
to  a  possible  basis  for  the  rough  measurement  of  proteoclastic  activity, 
since  the  water  soluble  protein  increased  with  length  of  extraction 
period,  at  the  expense  of  the  alcohol  soluble  protein.  In  the  second 
paper  Martin  (1920),  demonstrates  an  increase  of  gas  producing 
capacity,  e.  g.,  diastatic  power,  with  the  increasing  percentage  of  wet 
and  dry  gluten  as  the  source  of  the  flour  progressed  from  the  center 
of  the  endosperm  outward  to  the  cortex.  This  data  would  appear 
to  corroborate  the  opinion  of  Teichek  (1904),  that  diastase,  while 
largely  concentrated  in  the  germ,  is  also  distributed  throughout  the 
endosperm  and  extends  throughout  the  wheat  berry.  The  studies  of 
Whymper  (1909),  Mann  (1915),  and  others,  on  the  other  hand  point 
to  the  embryo  as  the  only  source  of  diastase  in  the  grain.  A  further 
examination  of  these  results,  with  analyses  of  both  diastatic  action 
and  nitrogen  partition  might  lend  support  to  the  postulate  that  the 
diastase  of  the  wheat  berry  is  associated  with,  or  held  by  the  protein 
body  and  functions  normally  only  in  their  presence.  Also  that  the 
peptizing  action  of  the  proteoclastic  enzymes  on  the  proteins  has 
some  bearing  oa  the  action  of  the  diastase. 

The  difficulty  of  using  the  expansion  volume  of  a  dough  as  a 
measure  of  diastatic  capacity  is  due  to  the  weakening  of  the  gluten 
after  the  second  or  third  hour.  "Curves  plotted  to  show  the  relation 
between  the  amounts  of  gas  generated  and  the  volumes  of  the  doughs 
were  fairly  regular  for  the  first  part  of  the  experiment,  but  erratic 
for  the  latter  part."  (Martin  (1920).  The  recent  work  of  Bailey 
and  Weigley  (1922)  afford  a  more  complete  summary  of  this  relation- 
ship. 

PRELIMINARY  DISCUSSION 
There  are  a  number  of  factors  v^ich  limit  the  production  of  maltose 
in  a  dough  by  the  agency  of  diastase. 

1.  Liquifying  and  Saccharogenic  Action.  —  Baker  and  Hulton 
(1908)  believed  that  the  diastase  of  grains  contained  both  a  liquifying 
and  a  saccharogenic  part  so  that  the  amyloclastic  power  would  become 
the  limiting  factor  in  maltose  production.  This  idea  is  frequently 
expressed  in  the  literature  on  diastase  in  bread  making,  but  to  date 
no  satisfactory  methods  have  been  developed  to  accurately  measure 
it.  Nor  has  that  phase  of  the  problem  been  undertaken  in  this  inves- 
tigation for  reasons  which  will  appear  further  on  in  this  discussion. 

17 


The  presence  of  these  two  distinct  activities  in  malt  and  wheat  dias- 
tase have  nevertheless  been  well  established. 

Without  attempting  to  analyze  the  numerous  but  inconclusive  data 
on  the  identity  of  the  two  distinct  activities  in  diastase,  it  is  sufficient 
to  recognize  their  interpendence  in  the  production  of  a  fermentable 
sugar  from  natural  starch,  and  summarize  the  later  work  on  the  sub- 
ject. Sherman  and  Schlesinger  (1913)  made  comparative  measure- 
ments of  the  amyloclastic  and  saccharogenic  power  of  the  malt  and 
pancreatic  preparations.  Their  paper  offers  a  comprehensive  list  of 
references  on  the  distinction  betw^een  the  tw^o  kinds  of  activity,  but 
no  conclusions  are  drawm  as  to  the  identity  of  the  separate  enzymes. 
While  their  results  on  pancreatic  amylase  indicate  a  fairly  constant 
relationship  between  the  amyloclastic  and  saccharongenic  activity  of 
their  pancreatic  preparations,  the  same  method  of  measurement  failed 
for  the  malt  preparations,  the  amount  of  starch  liquified  apparently 
being  less  than  the  maltose  produced  therefrom.  The  explanation 
suggested  to  account  for  the  observed  discrepancy  was:  That  these 
two  activities  are  characterized  by  different  conditions  of  optima  for 
their  respective  rates,  i.  e.,  of  temperature,  acidity,  and  salt  concen- 
tration. Sherman  and  Thomas  (1915)  supplied  further  evidence  in 
support  of  that  explanation  by  their  measurements  of  the  optima 
for  amyloclastic  and  saccharongenic  actions.  Whether  this  explana- 
tion be  the  correct  one,  and  the  two  distinct  activities  be  due  to 
separate,  conjunctive  enzymes;  or  whether  the  special  properties  of 
organic  colloidal  catalysts  can  perform  the  two  degrees  of  hydrolytic 
power  vmder  the  influence  of  changing  conditions  in  media ;  it  should 
be  possible  to  more  accurately  characterize  the  amyloclastic  and  sac- 
charogenic parts.  To  mention  only  a  few^  of  the  possibilities,  it  would 
appear  that  the  researches  of  Sorenson  (1917),  Robertson  (1920), 
Meutscheller  (1920),  and  Warden  (1921),  in  the  application  of  phys- 
ical and  electro  chemistry  to  biological  problems,  furnish  many  meth- 
ods which  might  be  applicable  to  the  separation  and  identification  of 
these  two  enzymes  if  they  exist  as  such,  or  to  the  establishment  of 
the  identity  of  the  catalyst  responsible  for  the  different  degrees  of 
hydrolysis.  The  remarkable  results  of  Warden  add  another  link  in 
the  chain  of  accumulating  evidence  for  the  hypothesis  that  enzymic 
activity  is  but  another  manifestation  of  the  catalytic  process  function- 
ing by  virtue  of  those  surface  forces  which  are  active  in  colloidal 
particles.  Such  studies  are  obviously  beyond  the  compass  of  this 
set  of  experiments  and  pertain  more  to  the  theory  of  enzyme  action. 

In  the  methods  of  Sherman  and  Schlesinger  noted  above,  soluble 
(gelatinized)  starch  w^as  again  used  for  the  measurement  of  both 
amyloclastic    and    saccharogenic    power,    and    therefore    cannot    be 

18 


applied  directly  to  the  question  of  amyloclastic  activity  as  the  limiting 
factor  in  diastatic  action  in  the  dough. 

2.  Resistance  of  Different  Starches  to  Diastatic  Action.  It  is  only 
the  final  production  of  maltose,  resulting  from  the  diastatic  activity 
of  a  given  flour,  which  is  of  consequence  to  the  baker,  and  the  various 
limiting  factors,  including  the  amyloclastic  activity  and  the  resistance 
of  that  particular  starch,  are  summarized  in  that  final  result.  Yet 
when  a  method  is  applied  also  to  the  measurement  of  diastatic  powers 
of  malt  preparations,  which  are  intended  to  act  on  the  natural  starches, 
the  question  of  the  resistance  of  the  natural  wheat  starches  becomes 
of  first  importance,  both  from  the  standpoint  of  the  final  product,  and 
for  the  selection  of  a  standard  substrate. 

There  appears  to  be  a  difference  in  the  condition  of  the  starch  in 
the  various  kinds  of  flour.  The  difference  was  noticed  by  us  in 
working  with  suspensions  of  the  flour  samples  under  investigation. 
Whymper  (1909)  by  microphoto  examination  of  starch,  showed  dif- 
ferences in  the  resistance  of  different  starch  granules  in  flour  to  dias- 
tase. Simpson  (1910)  "has  shown  that  under  certain  conditions  a 
small  proportion  of  flour  converted  into  sugar  a  quantity  of  ungelatin- 
ized  starch  equal  to  8%  of  the  weight  of  the  flour,  but  that  under 
identical  conditions  the  same  quantity  of  the  same  flour  converted 
a  quantity  equal  to  400%  of  its  own  weight  into  sugar  when  gelatin- 
ized starch  was  used."  Stone  (1897),  was  of  the  opinion  that  there 
were  differences  in  the  action  of  enzymes  on  starches  of  different 
origin,  but  Ford  (1904),  confirming  O'Sullivan's  previous  observa- 
tion (1904),  concluded  that  there  is  practically  no  difference  in  the 
action  of  diastase  on  starches  even  of  different  origin  under  com- 
parable conditions  of  acidity  and  temperature.  Their  data,  however, 
was  obtained  on  modified,  or  "soluble"  starch,  and  so  may  not  be 
applied  to  the  question  of  the  biological  differences  in  natural  wheat 
starch.  Sherman,  Walker,  and  Caldwell  (1919)  also  reached  the  con- 
clusion that  there  was  little  or  no  difference  between  the  resistance 
of  different  starches  to  the  same  diastase  preparation,  but  their 
starches  had  likewise  been  boiloH.  The  important  work  of  Reichert 
(1916),  and  of  Dox  and  Roark  (1917),  would  lead  one  to  expect  a 
variation  in  the  resistance  of  starches  of  different  origin  to  the  action 
of  enzymes.  Stakman  (1918)  and  Leach  (1919)  have  pointed  out 
the  significance  of  resistance  to  parasitism  which  is  shown  by  different 
varieties  of  wheat.  Considering  the  penetration  of  parasites  into  the 
host  as  the  result  of  enzymatic  action,  the  variable  resistance  of  dif- 
ferent biological  conditions  to  enzymes  may  well  be  expected  to 
show  in  the  starch  granules  as  well  as  in  other  plant  tissues.  The 
recent  articles  by   Samec  and   his  co-workers    (1921)    present   some 

19 


extremely  interesting  possibilities  in  the  chemical,  physical,  and  elec- 
trometric  differentiation  between  starches  of  different  biological 
origin.  But  the  susceptibility  of  biologically  different  samples  of 
wheat  starch  to  diastatic  activity  is  but  one  of  the  controlling  factors, 
and  like  the  other  factors,  is  reflected  in  the  resulting  degree  of  dia- 
static power. 

3.  Difference  Between  Autolytic  and  Extracted  Enzyme  Activity. 
In  the  wheat  berry  the  diastase  is  laid  down  by  the  plant  for  the 
purpose  of  transforming  the  starch  granules  of  the  endosperm  into 
soluble  sugar  available  for  assimilation  and  growth  of  the  young 
seedling.  Whether  or  not  in  the  economy  of  the  seed  there  is  a 
separate  preparatory  or  liquifying  enzyme,  a  cytase  which  prepares 
the  starch  granule  such  as  suggested  by  Armstrong  (1910),  and  makes 
it  susceptible  to  the  hydrolytic  action  of  the  diastase  (Wallerstein 
1917,  and  others),  the  result  is  the  same.  At  any  rate  during  panary 
fermentation,  the  diastase  of  the  flour,  and  likewise  any  diastase 
added  to  the  dough  in  the  form  of  malt  flour,  malt  extract,  or  other 
diastatic  preparation,  must  produce  maltose  from  the  wheat  starch 
as  it  exists  in  the  flour,  unless  other  forms  of  'starch  be  added  to  the 
dough. 

The  results  of  Ford  and  Guthrie  (1908),  have  indicated  that  the 
greater  enzymic  activity  was  obtained  by  auto-digestion  of  malt,  and 
by  addition  of  salts  and  proteoclastic  enzymes,  those  of  Baker  and 
Hulton  (1908)  that  flour  extracts  do  not  furnish  a  true  measure  of  the 
diastase  present  in  the  flour;  those  of  Sherman  and  Baker  (1916),  that 
purified  malt  extracts  show  different  activities  on  different  forms  of 
starch  substrate  (prepared),  and  those  of  Swanson  and  Calvin  (1913) 
have  demonstrated  the  value  of  the  autolytic  method  for  flours.  Yet 
the  methods  in  general  use  at  present  for  the  measurement  of  diastatic 
power  all  depend  upon  the  action  on  "soluble"  starch.  In  other 
words,  an  artificial  and  by  no  means  standard  substrate,  whose  col- 
loidal characteristics  have  been  profoundly  altered,  is  the  starting 
point  for  an  arbitrary  procedure,  the  result  of  which  often  has  no 
bearing  on  the  activity  intended  to  be  measured.  It  was  mentioned 
above  that  the  same  diastase  acting  on  soluble  or  gelatinized  starch 
may  produce  several  hundred  times  as  much  maltose  as  when  allowed 
to  act  on  raw  or  unbroken  starch  kernels.  It  will  be  demonstrated 
later,  (pp.  72,  table  XXII)  that  this  is  a  very  real  source  of  error  in 
the  determination  of  diastatic  power  of  different  diastase  preparations. 
Thatcher  and  Koch  also  found  that  extracts  prepared  by  their  method 
gave  lower  diastatic  values  than  were  obtained  in  equal  aliquots  of 
unfiltered  extracts  from  the  same  flour.  A  little  later  "attempts  to 
apply  the  method  in  a  comparative  study  of  the  diastatic  activity  of 

20 


wheat  flour  of  different  grades  and  various  processes  of  manufacture 
gave  the  surprising  result  that  extracts  of  approximately  uniform  dias- 
tatic  qualities  were  obtained  from  flours  of  widely  varying  character 
and  baking  qualities."*  Still  more  recently  R.  W.  Thatcher  and  Cor- 
nelia Kennedy  (1917)  obtained  some  valuable  data  bearing  on  the  loss 
of  diastatic  activity  in  flour  extracts  by  various  methods  of  treatment.** 
One  series  of  flour  samples  were  subjected  to  auto-digestion  with 
water  (1 :4)  for  1  hour  at  0°,  and  the  filtered  extract  allowed  to  act 
on  soluble  starch  at  different  temperatures,  as  in  the  proposed  method 
of  Thatcher  and  Koch  (1914).  The  following  table  shows  the  surpris- 
ing difference  in  result: 

TABLE  II. 

The  difference  in  diastatic  activity  between  auto-digestion  and  the 

action  of  extracted  diastase  on  soluble  starch. 

Cuprous  Oxide  Produced  by  Diastase 
from  .5  Grams  of  Flour 


•; 

Auto-digestion  of 

Extracted  1  hr.  @  0°C 

Flour-water  (1:4) 

Filtered   Extract  on 

Temperature  of 

1.  hour 

Soluble  Starch  1  hr. 

Action  on  Starch 

Grams 

Grams 

40° 

.02390 

.08485 

50° 

.05497 

.08125 

62' 

.15296 

.08050 

It  was  likewise  found  that  the  unfiltered  extract  acting  on  soluble 
starch  at  40,  50  and  62° C  showed  1.673  g.  Cu.  per  .5  g.  flour  as  com- 
pared to  only  .4302  g.  Cu.  per  .5  g.  flour  for  the  filtered  extract;  a  loss 
of  about  74%  of  its  activity  through  filtration.  An  examination  of 
the  residue  on  the  filter  would  indicate  that  the  tenacious  glutinous 
mass  had  adsorbed  some  of  the  enzyme,  or  else  had  held  back  an 
activator.  This  residue  was  apparently  not  tested  directly  for  its 
activity.  But  samples  of  gluten  were  washed  out  in  the  usual  manner 
and  wiere  kneaded  with  distilled  water  until  no  more  starch  could  be 
removed.  After  dispersion  in  N/200  lactic  acid  their  diastic  activity 
on  soluble  starch  was  compared  with  an  extract,  both  filtered  and 
unfiltered.     The  results  are  given  in  Table  III. 

TABLE  III. 

The  relative  diastatic  activity  of  water  extracts  and  of  gluten 

from  the  same  flour. 

1.  0.895  g.  Cu.  per  .5  g.  flour   (Filtered  extract  on  soluble  starch) 

2.  3.200  g.  Cu.  per  .5i  g.  flour  (Unfiltered  extract  on  soluble  stardh) 

3.  0.818  g.  Cu.  per  .5  g.  flour  (Gluten  dispersed  with  N/200  lactic) 


*(Geo.  P.  Koch's  unpublished  results  in  a  thesis  presented  to  the  Faculty 
of  the  Graduate  school  of  the  University  of  Minnesota,  in  partial  fulfilment 
of  the  requirements  for  the  degree  of  Master  of  Science). 

♦♦(Grateful  acknowledgment  is  hereby  made  to  these  authors  for  their 
permission  to  publish  the  results  of  their  experimental  data  in  this  connection). 

21 


It  would  thus  appear  that  the  gluten  had  retained  nearly  half 
of  the  diastatic  activity.  Unfortunately  there  is  no  data  available 
whereby  a  correction  can  be  applied  for  a  possible  activating  influence 
of  the  N/200  lactic  acid.  This  absorption  of  enzyme  by  gluten  was 
further  examined  by  extracting  4.  g.  flour  with  100  cc.  H^O,  (in  which 
were  dissolved  .006  g.  takadiastase)  for  1  hour  @  0°.  Part  of  the 
extract  was  filtered  and  compared  to  the  unfiltered  portion  in  its 
action  on  soluble  starch.  The  unfiltered  extract  produced  6.725  g. 
Cu.  as  against  only  3.215  g.  Cu.  for  the  filtered  extract.  Baker  and 
Hulton  have  found  and  recorded  that  some  diastatic  activity  w^as 
shown  by  the  glutens  thus  washed  out  of  wheat  flour. 

In  the  course  of  these  experiments  of  Thatcher  and  Kennedy  it  was 
desired  to  ascertain  whether  only  maltose  was  produced  by  diastasis. 
The  increase  in  Fehling  reduction  by  inversion  of  the  solution  after 
diastatic  action  had  been  stopped  was  measured. 

TABLE  IV. 

The  relative  reduction  of  Fehling  solution  before  and  after  inversion 

of  the  sugars  produced  by  diastatic  action. 

Before  Inversion  After  Inversion 
grams  cu.  grams  cu. 

I.     .02208  .04416 

II.     .03940  .06760 

III.  .02784  .05376 

IV.  .03264  .06192 

In  spite  of  the  evidence  of  Sherman  and  Plunnett  (1916),  fur  the 
production  of  small  amounts  of  glucose  in  addition  to  maltose  by  malt 
amylase,  the  fact  that  maltose  is  the  only  sugar  produced  by  diastase 
in  sufficient  quantity  to  be  of  any  significance  in  panary  fermentation 
has  been  recently  confirmed  in  this  laboratory.     (Collatz,  1922). 

One  peculiarity  of  the  results  obtained  by  Thatcher  and  Kennedy, 
however,  still  remains  to  be  explained.  The  diastase  activity  in  flour 
suspensions  has  been  shown  by  Sw^anson  and  Calvin,  and  confirmed 
here,  to  be  extremely  sensitive  to  temperature,  the  curve  appearing 
autocatalytic  in  character  up  to  nearly  60°C.  Yet  the  activity  of  the 
filtered  extracts  of  Thatcher  and  Kennedy  showed  the  same  value 
whether  acting  on  soluble  starch  at  40°,  at  50,  or  at  62°C,  while  the 
unfiltered  extract  showed  a  temperature  effect.  There  are  obviously 
several  possible  explanations:  (1)  The  filtration  may  remove  some 
substance  which  functions  as  an  activator  (Thatcher,  1921)  ;  (2) 
The  proteins  of  the  flour  may  absorb  some  essential  factor  in  the 
diastatic  action,  and;  (3)  the  contributing,  perhaps  controlling,  effect 
of  the  phosphates  and  other  buffer  salts  present  in  the  flour  (Bailey 
and  Collatz  1921,  and  Bailey  and  Peterson,  1921),  do  not  function 
under  those  conditions  of  extraction  and  measurement.     Neither  of 

22 


tUfse  explanaticms  seem  to  be  sufficient.  The  enzymes  are  un- 
doubtedly colloidal  in  character.  Their  hydrolytic  activity,  involved 
as  it  is  in  the  complex  properties  of  colloidal  material  such  as  surface 
action,  adsorption,  dispersion,  etc.,  is,  therefore,  extremely  sensitive 
tc  any  changes  in  the  electro-chemical  and  physical  nature  of  the 
media  in  which  they  are  acting. 

It  has  been  found  that  the  starch  as  washed  out  of  flour  carries 
considerable  diastase,  the  gluten  retaining  by  far  the  greater  portion 
Removal  of  the  protein  matter  from  the  starch  granules  by  dispersion 
and  repeated  washing  effects  the  removal  of  the  diastase,  indicating 
that  the  diastase  is  neither  adsorbed  to  nor  associated  with  the  starch. 
K.  Mohs  (1920,  1921),  has  presented  interesting  expositions  on  the 
colloidal  theory  of  diastatic  activity  and  though  probably  carried 
further  than  experimental  facts  warrant,  they  are  extremely  sugges- 
tive for  experimental  verification.  It  appears  certain,  in  short,  that 
extraction  of  diastase  from  biological  material,  with  subsequent  filtra- 
tion and  hydrolysis  of  modified  starch  by  the  filtrate,  will  not  afford 
an  accurate  conception  of  the  true  activity  as  it  exists  au  nature!. 
Therefore,  any  practical  method  for  the  measurement  of  the  value  of 
diastatic  enzymes  in  baking  must  be  based  upon  condlt'ons  as  they 
exist  in  the  dough.  The  increasing  general  use  of  diastatic  prepara- 
tions in  baking  practice  for  the  improvement  of  bread  makes  it  im- 
perative that  a  new  procedure  be  developed.  It  must  give  a  measure 
of  the  ability  of  that  diastatic  preparation  to  produce  sugars  which 
can  be  utilized  by  the  yeast  for  growth  and  carbon  dioxide  production. 

EXPERIMENTAL 

A  survey  of  the  literature  on  diastatic  activity  has  shown  the 
unquestionable  importance  of  these  enzymes  in  panary  fermentation. 
The  fact  that  the  bakers  in  America  are  estimated  to  use  something 
over  thirty  million  pounds  of  malt  per  year,  a  large  part  of  which  is 
diastatic  in  character,  with  a  probable  valuation  of  over  two  and  a 
half  million  dollars,  is  sufficient  evidence  of  the  economic  interest  in 
the  problem.  The  relative  impojrtance  of  this  activity  as  a  factor  in 
the  composite  strength  of  wheat  flours  has  not  been  settled.  The 
interest  of  the  baker  in  this  question  has  steadily  grown  until  a  satis- 
factory answer  should  contribute  materially  to  baking  science. 
Consequently  the  general  use  of  diastatic  preparations  for  the  im- 
provement in  volume  and  flavor  of  bread,  and  the  numerous  but  in- 
conclusive contributions  found  in  the  literature  on  the  effects  of  dias- 
tase in  panary  fermentation,  have  all  served  to  demonstrate  the  great 
desirability  of  further  research  on  the  diastatic  enzymes  of  wheat 
flour  in  their  relation  to  flour  strength. 

22> 


The  Materials.  To  obtain  data  wliich  would  furnish  the  basis  for 
conclusive  evidence  as  to  the  role  taken  by  diastase  in  dough 
fermentation,  it  was  considered  necessary  t  o  obtain  samples 
of  wheat  flours  of  widely  varying  characteristics.  Samples  of  flour, 
of  different  grades  and  baking  strengths  were  accordingly  obtained 
from  eight  of  the  typical  bread-flour  wheat  producing  areas  of  North 
America.  These  districts  include  the  Northern  Great  Plains  area,  the 
Washington  Walla  Walla,  the  Saskatchewan  and  Alberta  Canadian, 
the  Kansas,  the  Utah  irrigated,  the  Montana  Dry  Farming  and  the 
Ohio  wheat  areas.  The  Sitka,  Alaska  mills  were  not  yet  completed 
and  the  Alaskan  wheat  sample  obtained  was  too  small  to  furnish  suf- 
ficient standard  flour  for  comparison  with  the  other  samples.  The 
most  of  these  samples  of  flour,  in  fifty  pound  lots,  were  obtained  di- 
rect from  the  commercial  mill  in  which  they  were  ground.  In  the 
case  of  samples  Nos.  1012,  1013,  and  1014  the  wheat  was  obtained 
from  Fergus  county,  Montana,  and  w^as  selected  to  represent  the  aver- 
age wheat  as  raised  in  the  district.  .  It  w^as  shipped  direct  to  Fargo, 
N.  D.  and  there  reduced  to  flour  at  the  experimental  mill  operated  b> 
the  Agricultural  Experiment  Station  of  North  Dakota.  In  all  cases 
samples  of  the  wheat  from  which  the  flour  w^as  milled  were  kept  foi 
further  check  and  examination.  Thus  the  flours  studied  in  reference 
to  their  diastatic  activities  are  known  to  be  representative  of  the  wheat 
growing  districts  from  whence  they  came.  It  should  be  remarked, 
however,  that  three  of  the  samples,  number  1001,  1002  and  1009  are 
milled  from  blended  wheats.  Numbers  1001  and  1002  are  patent  and 
1st  clear  respectively,  milled  from  a  regular  "mill  mix"  and  are  typi- 
cal of  the  flours  generally  milled  in  that  locality.  Number  1009  should 
receive  special  mention  because  of  its  relation  to  the  other  flours.  It 
is  a  well  known  commercial  brand  of  high  grade  patent  flour,  milled 
from  a  two-wheat  blend  in  the  Northwest,  and  was  selected  because 
of  its  superior  quality.  The  composite  of  its  "strength"  characteris- 
tics, as  well  as  its  combination  of  qualities  as  shown  in  the  baked  loaf, 
gave  it  the  highest  baking  value  of  any  of  the  flours  available.  Con- 
sequently this  sample  was  used  as  a  standard  against  which  to  com- 
pare all  others. 

The  objection  might  be  raised  that  these  samples,  numbering  four- 
teen in  all,  do  not  furnish  sufficient  bases  of  seasonal  and  climatic  va- 
riation for  a  complete  characterization  of  enzymatic  activity  in  rela- 
tion to  strength.  Yet  it  is  believed  that  sufficiently  characteristic 
data  has  been  obtained  to  justify  certain  conclusions  and  to  furnish 
the  foundation  for  further  investigation  into  the  behavior  of  these 
biological  catalysts  of  wheat  flour. 

A  history  and  description  of  the  flour  samples  follows : 

24 


TABLE  V. 


History  and  Description  of  Flour  Samples. 


Sample 
No. 

1001 

1002 

1003 

1004 

1005 

1006 

1007 

1008 

1009 
1010 

1011 

1012 

1013 

1014 


Locality 
(Where  Grown) 


Type 
Turkey  Red 
Turkey  Red 
Little  Club 


-Wheat- 


Flour  Grade 


Reno  County 

Central  Kansas 

Reno  County 

Central  Kansas 

Washington 

Walla  Walla 

Red  River  Valley     Marquis  and 

North  Dakota  Bluestem 

Red  River  Valley      Marquis  and 

North  Dakota  Bluestem 

Canada  Marquis 

Southern  Alberta 

Canada  Marquis 

Southern  Alberta 

Canada,  Saskat.       Marquis 

Valley,  Saskatoon 

Red  River  Valley     Mostly  Marquis     Hard  Red  Spring  Patent 

Utah  Like  Sonora  Straight 

Irrigated  Valley       California 

Wheats 
Ohio  Resembles  Soft  Red  Winter    Long  Patent 

Williams  County      Fultzo-Mediter- 
ranean 

Common  Turkey  No.  2  Hard  Red      Patent 

Red  Winter 


Class 
Hard  Red  Winter  Patent 
Hard  Red  Winter  1st  Clear 
White  Club  Straight 

Hard  Red  Spring   2d  Clear 
Hard  Red  Spring   Patent 
Patent 
1st  Clear 


Selected  Hard 

Red  Spring 

Selected  Hard 

Red  Spring 

Hard  Red  Spring    Patent 


Montana,  Judith 
Basin,  Dry  Farm- 
ing 
Montana.  Judith 
Basin,  Dry  Farm- 
ing 
Montana,  Judith 
Basin,  Dry  Farm- 
ing 


Common  Turkey  No.  2  Hard  Red      1st  Clear 
Red  Winter 

Common  Turkey  No.  2  Hard  Red      2d  Clear 
Red  Winter 


Further  notations  on  the  special  characteristics  of  the  individual 
samples  will  be  recorded  in  connection  with  the  experimental  data. 

In  order  to  obtain  a  satisfactory  conception  of  the  relative  baking 
strength  of  these  flours  it  was  first  necessary  to  make  preliminary  bak- 
ing tests.  These  were  carried  out  by  the  baking  expert  of  the  Amer- 
ican Institute  of  Baking,  and  under  the  author's  constant  surveillance 
and  supervision.  This  baker  had  had  wide  experience  with  all  types 
of  flours  in  various  parts  of  the  country,  and  so  was  especially  well 
fitted  to  bake  and  judge  these  loaves.  The  same  individual  baked  all 
of  the  flours,  in  groups  of  four,  repeating  the  standard  and  one  of  the 
other  flours  for  comparison  with  each  succeeding  day's  bake.  The 
method  used  was  that  developed  for  a  standard  baking  practice  by 
this  Institute  for  its  service  and  research  departments.  The  purpose 
of  this  standard  baking  test  was  to  produce  a  loaf  under  carefully  con- 
trolled and  duplicable  conditions  which  should  approach  as  nearly  as 


25 


possible  to  those  obtaining  in  the  average  American  bake  shop.  It 
is  recognized,  of  course,  that  conditions  of  fermentation  in  a  small 
dough  batch  must  of  necessity  differ  somewhat  from  those  in  a  thous- 
and pound  dough.  Nevertheless,  it  has  been  found  that  this  differ- 
ence, due  to  what  the  baker  calls  "mass  action,"  can  be  largely  com- 
pensated for  by  a  proper  increase  in  the  amount  of  yeast  and  a  propor- 
tionate allowance  in  temperature.  Experience  has  shown  that  the 
behavior  of  a  particular  flour,  as  represented  by  the  fermentation,  and 
by  the  "score"  of  the  finished  loaf,  can  generally  be  taken  by  the  ex- 
perienced baker  as  a  measure  of  the  baking  quality  of  that  flour  when 
subjected  to  the  conditions  of  quantity  production. 

Baking  Test.  A  description  of  the  apparatus  used  in  the  baking 
tests  will  first  be  given  so  as  to  facilitate  the  discussion  of  the  pro- 
cedure. The  service  laboratory  was  equipped  with  a  ten  loaf,  electric- 
ally heated  Despatch  bake  oven,  equipped  with  mercury  and  record- 
ing thermometers,  and  piped  for  low  pressure  steam.  The  fermenta- 
tion box,  72x24x13  inches,  and  proofing  cabinet,  58x22x13  inches,  in- 
side measurements,  were  electrically  heated,  with  thermostatic  con- 
trol, and  the  proofing  cabinet  was  also  piped  with  low  pressure  steam 
for  controlling  the  humidity.  The  one  pound  pans  in  which  all  the 
loaves  in  this  series  were  baked  have  the  following  dimensions :  Top, 
eight  and  five-eighths  by  four  and  five-sixteenths  inches;  bottom, 
eight  and  one-eighth  by  three  and  three-fourths ;  heignt,  two  and  on^ 
half  inches.  A  small  Hobart  three  speed  mixer  fitted  with  a  two 
pound  bowl,  was  used  for  the  mixing  of  all  doughs. 

Formula.     The   standard   formula,  in   terms   of   a   one   pound   loaf 
dough,  is  as  follows: 

Grams  Per  Cent 

Flour     325.0  100.0 

Water    179.0*  55.0 

Sugar    lU.U  3.0 

Yeast   8.0  2.5 

Salt    5.0  1.5 

Lard *. 6.5  2.0 

History  and  Description  of  Flour  Samples. 

*The  amount  of  water  added  depends  upon  the  absorption  of  the  flour 
employed.  The  "absorption"  was  determined  in  the  conventional  manner,  by 
doughing  up  100  grams  of  the  flour  and  recording  the  number  of  cubic  centi- 
meters of  water  required  to  produce  a  dough  of  the  proper  consistency.  It  is 
a  well  known  fact  that  the  "absorption"  as  determined  in  this  manner  must 
often  be  changed,  depending  upon  whether  the  dough  stiffens,  or  slackens 
during  the  course  of  fermentation.  The  absorption  values  as  recorded  in  the 
subsequent  data  are  those  found  by  actual  fermentation  to  be  the  most  desirable 
for  the  proper  fermentation  of  each  particular  flour.  This  is  a  departure  from 
the  custom  which  has  usually  been  followed  in  comparative  bake  tests  as 
recorded  in  the  scientific  literature,  but  in  accordance  with  commercial  practice 
the  baker  was  instructed  to  modify  the  absorptions  and  fermentation  times 
in  such  a  way  as  to  produce  the  best  possible  loaf,  i.  e.  to  show  the  greatest 
strength  of  the  flour  without  the  use  of  any  additional  ingredients. 

26 


The  Baking  Procedure. —  Flour  samples  of  650  grams  each  (required 
for  a  two-loaf  dough)  were  weighed  out  into  ten  inch  mixing  bowls 
and  set  in  the  fermentation  cabinet  over  night  at  27°  centigrade.  200 
grams  of  sugar  and  100  grams  of  salt  w^ere  weighed  out  together,  dis- 
solved in  water,  and  made  up  to  a  volume  of  two  liters.  The  lard  w^as 
weighed  out  in  13  gram  portions  for  each  dough.  The  yeast  was  a 
part  of  the  supply  delivered  fresh  each  morning  for  use  in  the  baking 
school,  and  showed  unusual  uniformity  in  fermenting  abiltiy.  The 
}'east  was  cut  from  the  center  of  a  one  pound  cake,  and  160  grams  of 
this  were  weighed  out  thirty  minutes  before  mixing  the  doughs.  This 
yeast  was  suspended  in  water  in  a  one  liter  flask  at  a  temperature  of 
27  degrees.  By  the  use  of  these  solutions,  200  cc  of  sugar-salt  solu- 
tion furnished  the  required  2.5%  of  sugar  and  1.5%  of.  salt  for  each 
two  loaf  dough,  while  100  cc  of  the  yeast  suspension  contained  16 
grams  of  compressed  yeast. 

It  has  been  previously  found  that  the  20.  grams  sugar,  10.  grams 
salt,  and  16.  grams  of  yeast  for  each  dough  displaced  just  21.6  cc  of 
water.  Therefore  it  was  necessary  to  take  this  extra  volume  into  ac- 
count and  add  it  to  that  volume  of  liquid  as  calculated  from  the  ab- 
sorption. For  example,  flour  1008  with  an  absorption  of  60  should 
require  650X60=390.  cc  of  water,  so  in  addition  to  200  cc  of  sugar- 
salt  solution  and  100  cc  yeast  suspension,  there  would  be  required 
90-1-21.6=111.6  cc  more  water.  The  sugar-salt  solution  and  the  yeast 
suspension  were  both  brought  to  approximately  27°,  and  the  extra 
volume  of  water  could  be  warmed  or  cooled  as  a  convenient  control 
for  the  temperature  of  the  dough,  which  was  always  brought  out  of  the 
mixer  at  an  even  27° C.  A  half  degree  rise  in  temperature  was  allow,ed 
for  each  minute  of  mixing.  To  mix,  the  flour  was  transferred  to  the 
bowl  of  the  machine  mixer,  200  cc  of  sugar  solution,  and  100  cc  of  the 
well  shaken  yeast  suspension  were  then  added  from  rapid  flowing 
pipetts,  and  the  mixer  started  on  the  lowest  speed.  As  soon  as  the 
flour  had  all  been  taken  up  in  the  dough  the  lard  was  added  and  mix- 
ing continued  at  second  speed  to  the  end  of  the  second  minute.  The 
dough  was  then  cut  down  from  the  revolving  arm  and  mixed  with  the 
mixing  arm  turning  at  high  spe^d  for  another  minute.  In  this  same 
manner  each  dough  received  the  same  thorough  mixing  of  approxi- 
mately the  same  number  of  revolutions,  and  of  three  minutes  dura- 
tion. Each  dough  was  then  accurately  weighed,  set  in  lightly  greased 
bowls  fitted  w^ith  large  clock  glasses,  and  placed  in  the  fermentation 
cabinet  at  27°C  (80.6°F).  The  average  total  fermentation  time  was 
five  and  one-half  hours  from  mixing  to  baking,  from  the  time 
of  the  first  "punch."  The  dough  was  considered  to  be  ready  for  the 
first  working,  variously   termed   "turning,"  "cutting  over,"   "knead- 

27 


ing/'  "knocking  down,"  or  "punching,"  by  the  appearance  of  the 
dough  surface  when  indented  by  the  finger.  If  the  outer  edges  of  the 
indentation,  instead  of  filling  in  again,  should  show  a  tendency  to  sag 
down,  after  a  moment,  the  dough  was  considered  ready  for  the  first 
punch.  This  consisted  of  removing  the  dough  from  the  bowl,  knead- 
ing it  lightly  five  or  six  times  to  expel  most  of  the  gas,  and  setting 
again.  Considering  this  as  60%  of  the  total  fermentation  time,  the 
remaining  period  was  divided  into  approximately  28%  and  12%  of  the 
total  time,  the  second  punch  following  the  first  in  about  50  to  60  min- 
utes, and  the  dough  going  to  the  bench  for  rounding  after  a  third 
punch  about  25  to  30  minutes  later. 

The  method  of  handling  the  dough  from  the  mixing  to  the  oven  can 
best  be  illustrated  by  an  example.  Time  mixed  9:20  a.  m. — ready  for 
first  punch  at  11:40  a.  m.;  from  mix  to  first  punch  140  minutes. 
Taking  that  as  60%  of  total  time,  the  total  fermentation  period  should 
be  233  minutes.  Taking  28%  of  233,  or  65  mintues,  the  second  punch 
would  come  at  12:45  p.  m.,  and  the  third  at  1:13  p.  m.  The  third 
punch  is  in  reality  a  rounding  up  of  the  dough,  corresponding  to  the 
machine  rounder  in  the  modern  bakery.  After  being  rounded  up  the 
doughs  were  allowed  to  stand  on  the  bench  for  fifteen  minutes,  then 
moulded  into  loaves. 

In  these  test  bakes  the  two  loaves  were  carried  through  as  one  dough 
to  facilitate  handling  and  temperature  control.  The  doughs  were 
weighed  and  their  temperature  recorded  at  each  punch.  When  taken 
to  the  bench  for  rounding,  this  dough  was  divided  into  two  equal 
halves,  and  each  half  rounded  up  separately.  In  the  first  test  bake 
the  rounded  doughs  w^ere  moulded  into  loaves  by  hand,  but  in  the 
second  and  third  bakes  uniformity  of  grain  and  texture  was  obtained 
by  running  them  all  through  a  Thomson  machine  moulder. 

The  loaves  were  then  panned  in  separate  pans  and  placed  in  the 
proofing  cabinet  where  they  remained  at  a  temperature  of  32.5°C,  in 
an  atmosphere  nearly  saturated  with  moisture,  until  ready  to  go  into 
the  oven.  The  duration  of  the  proof  was  usually  55  to  60  minutes, 
depending  somewhat  on  the  flour,  and  the  height  to  w^hich  the  doughs 
were  allowed  to  rise  before  baking  was  that  which  was  found  by  ex- 
perience to  give  the  best  appearing  loaf  with  this  type  and  size  of  pan. 

When  the  loaves  were  ready  for  the  oven,  the  steam  was  turned  on 
to  furnish  a  moist  heat  and  delay  crusting,  and  allowed  to  remain  on 
for  the  first  three  minutes  the  loaves  were  in  the  oven.  The  tem- 
perature of  the  oven  was  so  adjusted  that  it  registered  435 °F  at  this 
point  in  the  baking.  Twenty  to  twenty-five  minutes'  baking  was  suf- 
ficient to  produce  a  well  baked-out  loaf  with  a  deep  golden-brown 
colored  crust. 

28 


The  loaves  were  weighed  directly  out  of  the  oven,  again  after  one 
hour,  and  a  third  time  at  the  end  of  eighteen  hours.  The  volume  of 
the  loaves  was  taken  at  this  time  with  the  Central  Scientific  Com- 
pany's volume  machine,  using  mustard  seed,  the  volumes  recorded 
having  been  repeatedly  checked  up  and  corrected  by  displacements  in 
water.  The  loaves  were  scored  and  "placed"  as  to  baking  value,  the 
resulting  relative  position  being  the  result  of  independent  judging  by 
three  experienced  practical  bakers.  The  numerical  baking  value 
assigned  to  each  flour  is  the  average  of  all  the  different  scorings  of 
three  trial  bakes,  consisting  of  two  loaves  for  each  flour  in  each  bake. 
The  first  series  of  baking  tests  wias  made  in  December,  1920.  The 
baking  on  each  loaf  was  repeated  two  or  three  times  with  slight  varia- 
tion in  fermentation  time  and  absorption  as  indicated  by  the  possible 
improvement  of  the  loaf.  The  second  test  bake  was  made  the  middle 
of  March,  1921  in  order  to  properly  classify  some  new  samples  just 
received.  The  order  of  baking  value  and  relative  strength  of  flours 
had  not  altered  appreciably  over  that  three  months'  period.  The  third 
baking  test  was  completed  in  November,  1921.  Only  the  data  for 
this  last  series  need  be  given  here,  since  the  relative  position  and  score 
of  the  baked  loaves  remained  the  same  over  the  six  months'  period. 
And  though  the  flour  had  aged  somewhat,  as  shown  by  the  natural  pH 
of  the  water  extract,  the  slight  change  in  "absorption"  and  slightly 
improved  baking  characteristics,  the  final  bakings  represent  very  vvell 
the  best  "strength"  characteristics  of  each  flour. 

Table  VI  is  a  record  of  the  comparative  baking  tests ;  the  final  score 

being  a  summary  of  points  according  to  the  American   Institute  of 

Baking  standard   (1922). 

TABLE  VI. 

Record  of  Comparative  Baking  Tests. 

Times Weights 

Fer- 

men-  Proof-  Dough 
Flour  Absorb-  tation  ing   at  Dough  Loaf   Loaf   Loaf  Vol- 
Sample  tion   Period  Period  Mix  M'lded  Hot   1  hr.  18  hrs.  umes    Score 

No.    %   min.   min.  grams  grams  grams  grams  grams  c  c 

1009  59   255   60   564   '518   457   448   435   2160   100 

1001  58   231    60   543    539         488   460   2010    99 
1008   60   203    53    542    535    500   492   464    2000    97 

1002  58   225   53   532   527   491        462    1880    95 

1012  59   225   54   537   527   489        460   1870    91 

1006  61  195  55  541  536  494  486  471  1735  91 
1005  59  187  65  536  531  496  484  464  1820  90 

1010  58  165  60  531  527  491  482  468  1760  83 

1011  56  229  51  529  524  489  479  459  1720  76 

1003  53    186    57    520        473   464   438    1650    6?> 

1013  58   224   51    528    532   496   487   468    1630    56 

1007  65    165   46   557   552   512   500   478   1460    46 

1004  58    192   45    549         504   498   473    1415    35 

1014  59   206   43   540      >   497   490   466   1295    32 

29 


Clarification.  The  chief  objection  to  the  application  of  autodiges- 
tion  for  the  measurement  of  diastatic  power  has  been  due  to  the  col- 
loidal character  of  the  flour-water  or  malt-water  suspensions.  The 
longer  the  digestion  continues  the  greater  the  degree  of  dispersion  of 
these  colloidal  protein  and  dextrin  products.  Considerably  difficulty 
has  been  encountered  in  obtaining  a  clear  solution  of  sugars  from 
enzymatic  action  and  sufficiently  free  from  those  colloids  which  inter- 
fere w4th  quantitative  sugar  determinations.  Lead  acetate  as  a  clar- 
ifying agent  for  flour  or  malt  solutions  is  exasperating  in  its  slowness 
and  poor  results.  It  was  found  to  be  practically  worthless  for  this 
work  because  of  its  failure  to  stop  diastatic  action. 

The  previous  inhibition  of  enzymatic  activity  by  acid  or  alkali  is 
not  satisfactory  because  of  the  necessity  of  again  neutralizing  the 
solution  before  adding  the  lead  reagent. 

The  first  obstacle  to  overcome  w^as,  therefore,  that  of  clarification. 
The  literature  supplies  numerous  methods  for  investigation.  Blish, 
(1918)  made  a  study  of  protein  precipitants  and  reported  that 
"reagents  ordinarily  used  for  precipitating  proteins,  such  as  alcohol, 
acetic  acid,  trichlor-acetic  acid,  salts  of  heavy  metals,  colloidal  iron, 
aluminum  hydroxide  cream,  phosphotungstic  acid,  and  tannic  acid, 
are  for  various  reasons  unsatisfactory  for  removing  gliadin  from  water 
extracts  of  flour."  He  recommended  tenth  normal  copper  sulfate  and 
sodium  hydroxide  as  the  most  efficient  precipitant  of  protein  nitro- 
gen. Phosphotungstic  acid  appeared  to  be  the  most  serviceable  for 
rapid  work,  and  was  the  reagent  used  by  Swanson  and  Calvin  (1913) 
and  by  Thatcher  and  Koch  (1914),  for  the  clarification  of  their  flour 
suspensions  and  extracts.  The  excessive  cost,  however,  prohibits  its 
use  in  quantity  for  control  and  service  laboratories  when  other  re- 
agents can  be  substituted.  Folin  and  Wu  (1919),  developed  a  new 
protein  precipitant,  tunstic  acid,  which  they  applied  to  the  precipita- 
tion of  blood  proteins.  After  the  addition  of  one  volume  of  ten  per- 
cent sodium  tungstate  (NagWO^  .2H2O.),  to  diluted  blood  serum 
they  added,  with  shaking,  one  volume  of  two-thirds  normal  sulfuric 
acid.  The  resulting  precipitate  was  in  such  form  that  it  could  be 
easily  centrifuged  and  filtered.  The  acid  is  intended  to  set  free  the 
whole  of  the  tungstic  acid  and  to  neutralize  the  carbonates  usually 
present  in  the  commercial  tungstate,  with  about  ten  percent  in  exces.-;. 
Because  of  the  efficiency  and  ease  of  application,  combined  with  a 
greatly  reduced  expense,  it  would  seem  that  this  reagent  deserves  a 
wider  application  in  the  clarification  of  colloidal  protein  suspensions 
than  it  enjoys  at  present.  A  few  trials  on  flour  suspensions,  extracts, 
and  malt  syrups  gave  promise  of  its  being  better  suited  to  these  than 
any  other  method  yet  employed. 

30 


Preliminary  experiments  showed  that  2.  cc  of  a  15%  sodium  tung- 
state  solution  w:ere  sufficient  for  the  soluble  proteins  in  5  grams  o. 
flour,  or  3.  cc  for  10  grams.  These  preliminary  trials  did  not  always 
result  in  a  good  clarification  even  though  the  equivalent  amounts  of 
2/3  N.  H2SO4,  as  suggested  by  Folin  and  Wu,  were  employed.  Some- 
times the  supertant  liquid  became  clear  almost  immediately,  the 
flocculated  proteins  settling  out  rapidly,  and  again  the  cloudiness  per- 
sisted after  a  half  hour's  centrifuging.  The  reason  was  not  far  to 
seek.  The  addition  of  the  sodium  tungstate  to  a  suspension  of  sound 
flour  in  water  produces  an  alkaline  reaction.  The  complete  precipita- 
tion of  the  proteins  from  a  colloidal  suspension  depends  upon  their 
adsorption  to  the  tungstate  ion  and  the  subsequent  precipitation  of  the 
coagulated  aggregate  by  throwing  the  hydrogen  ion  concentration 
over  sufficiently  far  to  the  acid  side  of  the  isolectric  point.  This  was 
accomplished  by  Folin  and  Wu  through  the  addition  of  2/3  N.HgSO^ 
in  quantity  which  neutralized  the  combined  alkalinity  of  the  tungstate 
and  blood  serum  with  a  slight  excess ;  but  in  the  case  of  flour  extracts 
the  higher  buffer  value  *  of  the  phosphate  and  other  salts  present  re- 
quires a  much  larger  excess  of  acid  to  produce  the  necessary  hydro- 
gen ion  concentration.  A  number  of  clarifications  were  obtained  in 
which  the  supernatant  liquid  became  as  clear  as  water  after  a  few 
minutes  centrifuging.  The  hydrogen  ion  concentration  of  these  solu- 
tions was  determined  electrometrically  and  they  were  found  to  have 
values  in  terms  of  pH  ranging  from  2.117  to  1.337.  **  The  slightly 
cloudy  liquids  from  the  unsatisfactory  clarifications  all  showed  pH 
values  of  2.67  or  above. 

These  and  subsequent  results  confirm  the  fact  that  the  success  of 
the  sodium  tungstate  clarification  depends  upon  proper  acidification 
of  the  NagWO^  -  protein  suspension  to  a  hydrogen  ion  concentration 
of  l.XlO-2  or  more,  corresponding  to  a  pH  of  2.0  or  less.  There  is 
no  danger  of  precipitating  the  colloidal  tungstic  acid  hydrate  even  with 
a  much  larger  concentration  of  acid.  Instead  of  the  2/3  HgSO^  of 
Folin  and  Wu,  or  the  1.  N.  acid  which  was  first  used  in  these  experi- 


*(The  term  "buffer"  was  introduced  by  Fernbach  and  Hubert  (Comptes 
rend.  Acad.  Sci.  131,293  (1900).  It  was  repeatedly  used  by  Sorenson 
(Comptes  rend,  du  Lab.  de  Carls.  8,53  (1909),  (Ergebnisse  der  Physiologic  12,- 
523  (1912),  by  Henderson  (Ergebnisse  der  Physiologie  8,254  (1909)  and  by 
Jenny  Hempel  (Comptes  rend,  du  Lab.  de  Carls.  13,1  (1917)  to  designate  the 
effect  of  various  salts  in  the  media  which  dissociate  upon  the  addition  of  acids 
or  alkalies  and  therefore  "use  up"  different  amounts  of  the  titrating  reagent 
before  significant  changes  are  brought  about  in  the  hydrogen  ion  concentration 
of  the  media.  The  importance  of  these  buffer  salts  in  wheat  flours  has  been 
emphasized  by  Bailey  and   Peterson    (Jour.   Ind.    Eng.    Chem.    13,916    (1921). 

**(A11  electrometric  determinations  were  made  with  the  Leeds  and 
Northrup  Type  K  Potentiometer,  using  the  Bailey  (1920)  hydrogen  electrode. 
The  pH  values  corresponding  to  the  millivolt  readings  were  taken  from  the 
Schmidt  and  Hoagland  tables.) 

51 


ments,  concentrated  sulphuric  acid  added  from  a  micro-pipette  ap- 
pears to  be  more  efficient  in  precipitating  the  protein-tungstate.  The 
precaution  of  adding  the  acid  slowly,  drop  by  drop,  with  shaking  of 
the  solution  must  be  observed,  otherwise  a  local  concentration  will 
produce  a  precipitation  of  flocks  of  colloidal  tungstic  acid,  and  with 
further  danger  of  decomposing  some  of  the  carbohydrates.  Thymol 
Blue  (Clark  1920)  with  its  acid  range  at  pH  2.+  serves  as  a  very  con- 
venient indicator  for  the  first  few  trials  with  any  new  sample,  since  it 
is  necessary  to  add  only  two  or  three  drops  of  the  concentrated  acid 
in  excess  of  the  pink  color  to  produce  the  proper  acidity  for  com- 
plete precipitation.  After  a  few  trials  uniform  results  were  always 
obtained  by  measuring  the  acid  from  a  1.  cc  micropipette  or  counting 
the  number  of  drops  required  to  produce  the  proper  color  by  Thymol 
Blue. 

Because  of  their  higher  buffer  value  malt  flour  suspensions  are 
found  to  require  a  slightly  larger  amount  of  acid.  In  this  connection 
a  difficulty  was  later  encountered  in  the  clarification  of  suspensions 
to  which  considerable  quantities  of  acid  had  already  been  added.  The 
same  principle  of  colloidal  protein  precipitation  applies  here  as  well, 
and  the  clarification  is  satisfactory  if  the  suspension  is  first  neutral- 
ized by  means  of  a  few  drops  of  strong  NaOH.  The  Thymol  Blue, 
alkaline  range,  blue  color  (pH  8.0  to  9.6)  likewise  serves  for  this  point, 
and  though  the  hydroxyl  ion  concentration  does  not  need  to  be  carried 
so  far,  it  does  no  harm  as  the  subsequent  addition  of  acid  brings  it 
back  immediately  to  a  pH  of  2.0  or  less. 

The  suspensions  to  be  clarified  in  this  series  were  always  centrifuged 
for  two  or  three  minutes  to  save  time,  and  more  especially  to  form  a 
compact  mass  of  flour  solids  in  the  bottom  of  the  centrifuge  tube  from 
which  the  clear  supernatant  liquid  could  be  poured  or  pipetted  without 
stirring  up  any  of  the  material  which  had  been  thrown  down.  But  if 
a  centrifuge' is  not  available  the  starch  and  precipitated  proteins  settle 
out  clear  in  about  five  minutes,  and  if  desired  the  supernatant  liquid 
can  be  rapidly  filtered  through  a  fine  quantitative  filter  paper.  This 
is  especially  true  of  the  clarified  dilute  solutions  of  malt  extracts  in- 
tended for  sugar  determinations.  As  will  be  shown  later,  however, 
the  sodium  tungstate  clarification  followed  by  a  few  minutes  centri- 
fuging  effects  the  elimination  of  all  filtrations,  which  heretofore  have 
required  hours,  and  which  because  of  the  errors  thus  introduced  have 
been  the  stumbling  block  in  many  investigations  of  this  nature. 

The  amount  of  soluble  nitrogen  remaining  in  solution  was  deter- 
mined by  the  Kjeldahl  method  before  and  after  clarification,  using 
different  amounts  of  tungstate  and  varying  acidities.  The  residual 
nitrogen  appears  to  reach  a  fairly  constant  minimum  for  the  flour  used 

32 


under  the  conditions  of  clarification  described  above.  Different  ma- 
terials show  different  content  of  solublie  amino  nitrogen  and  ammonia 
nitrogen  which  is  not  removed  by  the  tungstate  procedure,  but  which 
shows  no  vitiating  effect  on  the  reducing  sugar  determinations.  The 
results  of  the  above  discussion  are  summarized  in  table  VII,  which 
shows  the  nitrogen  remaining  in  solution  after  treatment  with  sodium 
tungstate. 


TABLE  VII. 

The  efficiency  of  the   Sodium   Tungstate  reagent   as  a  clarifying 

agent   for  flour   suspensions   at   various 

concentrations  and  acidities. 


Flour 
grams 

5 

Final 

Volume  of 

Clarified 

Solution 

cc. 

100 

Na^WO 

15% 

cc. 

4 

Acid  Added 

5.  cc^^^NH.SCX 

pHof 
•esulting 
Solution 

3.88 

Nitrogen 

Remain- 
ing in 

100 
cc's  of      Remarks  on 

Solution    Clarification 
grams 
.0034     Fair 

5 

100 

2 

2.5  cc  N/1  H.SO« 
10  drops  in  excess 
by  Thymol  Blue* 

2.603 

.0021 

Excellent 

5 

100 

3 

Acid    by    Methyl 
Orange 

5.04 

.0129 

Poor;   very 
cloudy 

5 

100 

2 

10  drops  in  excess 
by  M.  O. 

3.446 

.0027 

Good 

5 

100 

2 

Acid  by  Thymol 
Blue* 

2.536 

.0021 

Very  good 

10 

200 

3 

4.  cc  N/1  H2SO4 

2.117 

.0026 

Excellent 

10 

200 

3 

.4  cc  Cone.  UzSOa 

1.468 

.0021 

Excellent  after 
1  hr.  digestion 

10 

200 

3 

.4  cc  Cone.  H,SO« 

1.457 

.0021 

Excellent  after 
3  hrs.  digestion 

10 

200 

0 

0.  cc 

5.778 

.0241 

JSfo  clarification 
after  1  hour  di- 
gestion. 

10 

200 

0 

0.  cc 

5.596 

.0379 

fTo  clarification 
after  3  hours 
digestion 

*(7.  drops  Thymol  Blue  Indicator). 


33 


Reducing  Sugars.  Several  methods  present  themselves  for  the  de- 
termination of  reducing  sugars  in  the  clarified  solutions  from  flour, 
or  malt  enzyme  digestions,  and  different  ones  have  found  favor  with 
the  different  workers  on  the  products  of  diastatic  action.  Swanson 
and  Calvin  (1913)  Thatcher  and  Koch  (1914),  and  Thatcher  and  Ken- 
nedy (1917),  applied  the  iodine  titration  method  to  the  residual  copper, 
after  the  Fehlings  reduction,  basing  their  work  on  the  articles  pub- 
lished by  A.  W.  Peters  (1912).  Spoehr  (1919)  modified  the  details  of 
the  sartie  method  for  application  to  small  amounts  of  sugar-containing 
juices  from  cacti.  He  found  that  his  solutions  contained  other  sub- 
stances than  sugar  which  contaminated  the  w^eight  of  reduced  cuprous 
oxide  and  so  made  it  necessary  to  determine  the  unreduced  copper. 
Harter  (1921)  followed  the  Clark  (1918)  modification  of  the  Scales 
(1915)  procedure  to  measure  the  reducing  sugars  produced  by  the 
diastase  of  Rhizopus  tritici.  The  method  of  Bertrand  (1910),  which 
determines  the  amount  of  copper  reduced  by  the  sugars,  has  been  but 
little  used  in  this  sort  of  work.  The  Bertrand  titration  method  fur- 
nishes a  convenient  way  of  checking  the  weights  of  the  precipitated 
cuprous  oxide,  and  was  so  used  in  this  laboratory  to  determine  the  ac- 
curacy of  the  crucible  weights  as  compared  to  the  actual  copper  re- 
duced. In  this  way  it  was  found  that  the  supernatant  liquids  from  the 
tungstate  clarification  as  carried  out  in  the  present  investigation  gave 
accurate  and  concordant  results  by  weighing  the  filtered  cuprous  oxide 
precipitate,  and  the  difficulty  in  contamination  of  the  reduced  copper 
by  non-sugars,  as  recorded  by  Spoehr,  was  therefore  not  encountered. 

Because  of  the  conditions  under  w-hich  this  work  was  done,  it  was 
found  more  convenient  to  use  the  prepared  gooch  crucibles  with  asbes- 
tos mats  for  filtering  the  reduced  copper  than  to  apply  the  iodine 
method  of  titration,  with  its  consequent  necessity  for  the  preparation 
of  solutions.  The  total  time  required  for  the  preparation  and  weigh- 
ing of  the  gooch  crucibles  is  hardly  greater  than  that  for  the  titration 
procedure.  Furthermore,  the  excellent  contribution  of  Shaffer  and 
Hartman  (1921)  has  shown  that  the  iodometric  titration  method  for 
residual  copper  as  previously  carried  out  is  not  without  its  possibili- 
ties of  serious  error.  On  the  other  hand  Quisumbing  and  Thomas 
(1921)  in  their  recently  published  article  on  the  reduction  of  Fehling 
solution  by  different  sugars,  point  out  several  possible  sources  of  er- 
ror in  the  reducing  sugar  determinations  according  to  the  official  Mun- 
son-Walker  procedure.  Unfortunately  these  two  articles  did  not  ap- 
pear until  the  work  reported  here  was  well  along  toward  completion, 
and  it  was  believed  best  to  complete  the  determinations  using  the  same 
comparative  procedure;  consequently  all  reducing  sugar  determina- 
tions reported  in  the  experimental  part  of  this  paper  were  made  by 

34 


the  Munson-Walker  method,  (A.  O.  A.  C.  Methods  of  Analysis,  re- 
vised, 1919.  Section  VII.)  A  battery  of  forty  No.  0  porcelain  gooch 
crucibles  were  used,  and  the  cuprous  oxide  resulting  from  the  Feh- 
lings  reduction  was  filtered  onto  thick  asbestos  mats,  washed,  dried 
and  weighed.  The  corresponding  weights  of  maltose  were  taken  from 
the  Munson-Walker  tables.  The  crucibles  were  fitted  with  mats  of 
properly  prepared  washed  and  ignited  asbestos,  at  least  1  cm.  in  thick- 
ness, and  washed  into  place  by  whirling  with  a  stream  of  hot  water 
from  a  wash  bottle.  They  were  then  washed  with  95%  alcohol  and 
dried  at  100°  for  1  hour.  The  weights  of  the  crucibles  so  prepared 
remained  constant  in  the  dessicator  for  periods  of  weeks. 

The  Fehling  solutions  used  showed  no  auto-reduction  in  the  blanks. 
The  only  standardization  necessary  was  to  run  a  few  blank  determina- 
tions in  order  to  apply  a  correction  for  each  new  lot  of  asbestos  pre- 
pared. The'  400  cc  Pyrex  beakers  used  for  Fehling  solution  reduc- 
tions were  of  approximately  uniform  thickness,  the  watch  glasses  used 
for  covers  fitting  rather  closely.  The  flame  was  so  adjusted  that  the 
different  samples  started  boiling  usually  within  five  seconds  of  the  four 
minute  period  stipulated.  The  chief  source  of  error  was  found  to  be 
in  the  loss  of  the  weight  show^n  by  different  lots  of  prepared  asbestos 
upon  pouring  the  hot  Fehling  solution  through  the  mats.  The  maxi- 
mum variation  seldom  w^ent  beyond  1  milligram  and  with  triplicate 
determinations  the  weights  of  Cuprous  oxide  usually  checked  within 
.2  to  .3  milligrams  of  the  average.  It  would  appear  highly  desirable 
for  this  sort  of  work  to  combine  the  method  of  Shaffer  and  Hartmann, 
and  of  Quisumbing  and  Thomas  into  a  standard  method  in  which  the 
sources  of  uncontrolled  error  are  reduced  to  a  minimum.  Also  the  ap- 
plication of  sodium  tungstate  as  a  clarifying  agent  for  protein-contair- 
ing  solutions  could  be  profitably  studied  in  its  connection  with  such 
a  method. 

Effect  of  Clarifying  Agent  on  Sugar  Determinations.  The  next 
point  requiring  investigation  was  the  effect,  if  any,  of  various  amounts 
of  the  sodium  tunt<state,  as  used  for  clarification,  on  the  determina- 
tion of  reducing  sugars  by  FeWing  solution.  Preliminary  trials  on 
flour-water  suspensions  with  and  without  added  dextro-«e  indicated 
that  the  addition  of  the  sodium  tungstate  in  excess  for  clarification  did 
not  affect  the  Fehling^s  reduction.  To  confirm  this  point  and  to  see 
what  results  could  be  obtained  from  unclarified  solutions,  the  follow- 
ing method  was  used :  five  grams  of  a  flour  showing  considerable  dia- 
static  activity  were  weighed  into  250  cc  beakers,  50  cc  of  water  at  27*'C 
were  added,  and  the  mixture  thoroughly  stirred.  In  some  cases  40  cc  of 
HgO  and  10  cc  of  a  0.5%  dextrose  solution  were  added  in  place  of  the 
water  alone.     These  were  allowed  to  digest  for  one  hour  at  27°C,  stir- 

35 


ring  at  intervals.  Some  of  the  samples  were  then  clarified  by  Na.2W04, 
using  Thymol  Blue  as  indicator.  In  the  other  samples  the  enzymic 
activity  was  inhibited  by  5.  cc  of  0.2N  NaOH.  After  clarification,  or 
inhibition  of  the  enzymic  action,  the  solution  was  diluted  to  100  cc  in 
a  volumetric  flask,  poured  into  a  100  cc  centrifuge  tube  and  whirled 
ten  minutes.  50  cc  of  the  clear  supernatant  liquid  were  thern  pipetted 
out  into  a  400  cc  beaker  for  determination  of  sugars  by  the  Munson- 
Walker  method. 

The  results  are  compiled  in  table  VIII  under  three  groupings, 
samplesnumbered  1  to  4  include  the  blanks,  5  to  7  the  flour  with  no 
added  dextrose,  and  numbers  8  to  13  the  flour  samples  with  a  known 
weight  of  dextrose  added. 

TABLE  VIII. 

The    determination   of    reducing    sugars    in    solutions    w^ith    and 
without  clarification  by  sodium  tungstate. 


Sample 
Number 


Flour 

grams 

0. 


15%  Na^WO. 
Dextrose  For 

Added     Clarification 
milligrams     cc. 
0.  0. 


4. 


0. 


50. 
0. 

50. 


7. 

5. 

0. 

8. 

5. 

50. 

9. 

5. 

50. 

10. 

5. 

50. 

11. 

5. 

50. 

12. 

5. 

50. 

13. 

5. 

50. 

10. 
2. 


Cu.O 
Veiffhed 

Average 
Weight 
of  Cu,0 

Expressed 

as  Dextrose 

Recovered 

grams 
-.0005 
-.0006 

grams 
-.0005 

milligrams 
none 

.1146 
.1150 

.1148 

49.7 

-.0004 
-.0002 
-.0006 

-.0004 

none 

.1138 
.1156 
.1140 
.1145 

.1145 

49.6 

.0800 
.0788 
.0795 

.0794 

34.0 

.0795 
.0782 
.0795 
.0800 

.0793 

34.0 

.01665* 

.01665 

6.8 

.2008 
.2004 

.2006 

89.0 

.1985 
.1987 

.1986 

88.6 

.1997 
.2004 
.1963 
.1963 

.1982 

88.4 

.2013 

.2013 

89.4 

.2027 

.2027 

90.4 

.1975 

.1984 

.1980** 

88.0 

♦Average  of  5  determinations. 
*♦  (Filtered) 


36 


The  values  recorded  as  sample  number  7  are  the  average  found  for 
live  determinations  of  the  natural  reducing  value  of  the  flour,  the  dias- 
tase having  been  inhibited  by  preliminary  tungstate  and  acid  treat- 
ment. 

The  determinations  recorded  in  Table  VIII  were  obtained  under 
conditions  of  poor  temperature  control.  The  De  Khotinsky  electric- 
ally heated  constant  temperature  water  bath  with  electrostatic  control 
which  was  used  for  all  subsequent  work  had  not  yet  been  installed, 
and  these  digestions  were  made  at  27° C  in  an  incubator.  The  tem- 
perature fluctuated  rather  widely  (around  .5  degree),  and  the  conse- 
quent variation  in  diastatic  activity  was  to  have  been  expected.  This 
series  was  not  repeated  as  such,  because  later  data  obtained  in  con- 
nection with  other  experiments  has  shown  that  the  conclusions  as  in- 
dicated by  Table  VIII  are  valid,  namely;  (1)  The  use  of  15%  sodium 
tungstate  in  quantities  up  to  5  cc,  and  sulfuric  acid  for  clarification  of 
flour  suspensions,  neither  interferes  with,  nor  affects  the  determina- 
tion of  reducing  sugars  in  the  clarified  solution  by  the  Munson-Walker 
method.  (2)  It  is  not  absolutely  necessary  to  clarify  the  supernat- 
ant liquid  obtained  from  the  centrifuging  of  a  5%  flour  and  water  sus- 
pension. This  should  be  further  qualified  by  notes  on  the  appearance 
of  the  Fehling's  reduction.  It  was  shown  in  every  case  that  clarifica- 
tion was  an  advantage  in  the  reduction  of  Fehling's  solution,  espec- 
ially in  the  case  of  cloudy  solutions.  If  unclarified,  the  boiling  Feh- 
ling  solution  foamed  badly,  and  the  formation  of  CugO  appeared  to  be 
hastened  by  the  coagulated  protein,  resulting  in  a  dark  and  somewhat 
muddy  looking  precipitate  which  filtered  badly,  and  showed  a  ten- 
dency to  adhere  to  the  surface  of  the  beaker.  (3)  The  use  of  the 
sodium  tungstate  clarifying  reagent  renders  the  solution  clear  and 
protein-free,  and  when  centrifuged  to  throw  down  suspended  matter 
it  eliminates  all  necessity  for  filtration.  Further  proof  that  filtration 
of  the  clarified  centrifugate  is  entirely  unnecessary,  is  evident  from 
data  obtained  in  other  experiments,  at  different  times  and  for  differ- 
ent purposes,  but  using  the  same  flour  sample  and  method.  The  ob- 
ject in  view  when  these  samples  were  run  bore  no  intentional  relation 
to  conclusion  (3),  but  the  resuUs  substantiate  those  obtained  in  table 
VIII.     This  data  is  collected  and  tabulated  in  Table  IX. 

TABLE  IX. 

The  determination  of  reducing  sugars  in  the  centrifugate  from  the 

sodium  tungstate  clarification  before  and  after  filtering. 

Sample  Grams  CuaO 

1  Filtered  .12591 

2  Filtered  .1259  >   .1256  Average 

3  Filtered  .1252J 

4  Unfiltered  .12601 

5  Unfiltered  .1251  \    .1255   Average 

6  Unfiltered  .1256J 

37 


On  the  other  hand,  a  very  few  trials  are  sufficient  to  convince  one 
that  filtration  of  the  unclarified  solution  from  a  flour-water  suspen- 
sion is  a  most  unsatisfactory  procedure  and  should  be  expected  to 
give  inaccurate  results  for  reducing  sugars. 

A  further  careful  scrutiny  of  the  data  in  table  VIII  brought  out  a 
peculiarity  in  the  determinations  which  has  not  yet  been  satisfactorily 
explained.  Comparing  samples  Nos.  5,  6  and  7,  and  Nos.  1,  4  and  7, 
it  appears  that  there  is  an  increase  of  reducing  sugars  as  determined 
by  weighing  the  Cu^O  over  and  above  that  added  in  the  form  of  dex- 
trose. This  question,  like  several  others  which  were  brought  out  in 
the  course  of  this  series  of  investigations,  is  recorded  here  with  what- 
ever explanation  appears  possible  from  the  data  at  hand,  with  the 
hope  of  enlisting  the  interest  and  experimental  efforts  of  other  work- 
ers. The  most  obvious  explanation  is  that  the  natural  reducing  value 
of  the  flour  extract  is  due  (probably)  to  dextrose,  while  the  increase 
of  reducing  power  after  one  hour  of  diastatic  activity  is  due  to  mal- 
tose. In  table  VIII  the  weights  of  CuaO  as  recorded  are  produced  in 
some  instances  by  dextrose  alon€  (samples  numbered  1  to  7  inclusive), 
and  in  others  by  maltose  alone  or  by  both  dextrose  and  maltose 
(samples  8  to  13  inclusive). 

TABLE  X. 

The  relation  of  actual  to  calculated  values  for  the  reducing  power 

of  dextrose  added  to  autolytic  digestion  of  diastatic  flour. 

(Data  taken  from  Table  VII). 

A  B 
In  ter^s  of     In  termig  of  Dextrose 
Cuprous  Oxide        and  Maltose 
Without  added  dextrose                      Milligrams 
Total  reduction  after  1  hour's  diastasis  (sam- 
ples Nos.  5  and  6) 79.35 

Blank  (natural  reducing  power  of  flour  (sam- 
ple No.  7) 16.65 

Difference  due   to  diastasis 62.70 

With  added  Dextrose 

Dextrose  added  (sample  Nos,  2  and  4) 114.58 

Blank  (natural  reducing  power  of  flour  (sam- 
ple No.  7) 16.65 


Total,  due  to  Dextrose 131.23 

Total  reduction  after  1.  hour  diastasis   (sam- 
ples Nos.  8  to  10  inclusively) 199.51 

Total  reduction  due  to  Dextrose 131.23 


Difference  due  to  diastasis 68.28 


Value   found    62.70 

Value  calculated 68.28 

Increase    5.58  mg.  2.96  mg. 

38 


Milligrams 

60.88  = 

■  Maltose 

6.86  = 

:  Dextrose 

54.02  = 

:  Maltose 

49.83  = 

:  Dextrose 

6.86  = 

:  Dextrose 

56.69  = 

:  Dextrose 

56.69  = 

=  Dextrose 

52.12  = 

=  Maltose 

108.81  = 

108.81 
110.77 

:  Total 
reducing 
sugar 

In  Table  X  the  data  has  been  rearranged  to  show  that  the  increased 
weight  of  cuprous  oxide,  in  the  range  of  values  between  63  and  131 
milligrams,  is  equivalent  to  a  positive  error  of  5.58  milligrams  of 
dextrose,  or  an  increase  of  nearly  9%  over  the  calculated  value.  Since 
the  average  error  of  the  method,  due  to  slight  differences  in  tempera- 
ture, volumes  and  weights  is  about  0.8  milligram  of  CugO,  the  above 
variation  is  more  likely  due  to  other  causes.  When  the  data  is  recal- 
culated into  terms  of  dextrose  and  maltose  respectively,  as  showm 
under  Part  B  of  Table  X,  the  error  is  reduced  somewhat,  due  to  the 
smaller  numerical  value  of  the  corresponding  reducing  sugars.  This 
method  of  calculation  gives  a  difference  of  approximately  3.  milli- 
grams of  reducing  sugars,  or  about  5.  per  cent.  Differences  of  similar 
magnitude,  and  positive  in  sign,  have  been  obtained  in  subsequent 
work. 

Adsorption  by  Starch.     The  next  explanation  to  suggest  itself  is  a 

displacement   concentration   due   to   the  volume  occupied   by   the   5. 

grams  of  flour  in  the  100  cc  flask.     It  wall  be  shown  that  this  accounts 

for  only  part  of  the  error.     The  hydrated  colloids  of  the  flour,  e.  g. 

starch  and  gluten,  might  be  expected  to  adsorb  some  of  the  sugars, 

thus  reducing  the  concentration  of  the     supernatant     liquid.     Thus 

there  would  exist  a  ratio  between  the  reduction  in  the  concentration 

of  sugars  due  to  adsorption,  and  the  increase  of  concentration  due  to 

volume  displaced  by  the  flour.     The  difference  then  in  result  between 

the  dextrose  recovered,  and  the  dextrose  added  to  the  flour  suspension 

plus  the  reducing  value  of  flour-water  suspension,  would  be  resultant 

Displacement  ^    ^. 

of  this  ratio  of-—^^ -. x  concentration. 

Absorption 

We  should  also  expect  these  colloids  to  be  sensitive  to  changes  of 
electrolytes  or  of  hydrogen  ion  concentration,  yet  the  results  of  sever- 
al trials  did  not  show  sufficient  adsorption  of  dextrose  by  flour  colloids 
in  excess  water  at  a  pH  of  6  or  less  to  be  recognized  by  the  methods 
employed.  On  the  other  hand,  the  errors  were  always  in  the  opposite 
direction,  that  is,  there  was  always  an  increase  in  sugars  recovered 
over  that  expected. 

Samples  of  both  wheat  flour 'and  wheat  starch  were  weighed  into 
accurately  graduated  flasks,  the  flasks  fiilled  to  the  mark  with  distilled 
water,  and  allowed  to  stand  either  one  hour  or  24  hours,  before  being 
brought  again  to  volume  and  weighed.  From  these  weights  it  was 
calculated  that  .5601  cc  of  water  is  displaced  per  gram  of  flour,  and 
.7086  cc  per  gram  of  starch.  Using  these  values  to  calculate  the  dis- 
placement concentration,  the  total  increase  in  weight  of  the  CuaO, 
due  to  increased  concentration,  should  be  approximately  2.2  milli- 
grams.    This  is  less  than  half  the  difference  actually  obtained.     For 

39 


want  of  a  better  explanation  the  remaining  error  might  be  laid  to  the 
increased  reduction  of  the  Fehling  solution  by  the  addition  of  dextrose 
to  the  maltose  in  solution.  Such  a  probability  is  suggested  by  the 
recently  published  work  of  Quisumbing  and  Thomas  (1921). 

Preparation  of  Wheat  Starch.  This  increased  recovery  of  reducing 
sugars  by  Fehling's  reduction  is  likewise  shown  by  some  of  the  data 
obtained  on  starch.  To  obtain  some  idea  of  the  behavior  of  starch 
in  flour  under  the  conditions  of  these  experiments,  several  samples  of 
wheat  starch  were  washed  out  of  flour  with  running  water,  the  gluten 
being  held  back  by  manipulating  over  a  No.  10  flour  silk  gauze. 
Repeated  washing,  decantation,  and  differential  centrifuging  failed 
to  remove  all  the  protein  matter  from  the  starch,  which  still  showed 
some  slight  diastatic  activity  after  driying.  Other  samples  were 
then  prepared  and  the  proteins  removed  by  dispersion  and  wash- 
ing with  very  dilute  NaOH.  Too  strong  soda  solution  gelatinizes  the 
starch,  rupturing  the  granules  and  even  though  a  concentrated  alcohol 
treatment  retrogrades  it  back  to  insoluble  starch  (Herzfeld  and  Kling- 
er,  1921.),  the  physical  and  chemical  character  of  the  material  has  been 
changed,  and  it  is  no  longer  comparable  to  the  raw  wheat  starch  of 
the  flour.  After  the  proteins  and  NaOH  have  been  washed  out  with 
distilled  water,  the  starch  is  carefully  centrifuged,  and  only  the  center 
portion  of  the  column  deposited  in  the  bottom  of  the  centrifuge  tube  is 
taken.  This  is  repeatedly  washed  with  cold  distilled  water  containing 
a  few  drops  of  HCl  per  liter  until  no  more  acid  is  removed  from  the 
solution.  The  material  is  then  filtered  on  a  Buchner  funnel,  washed 
with  re-distilled  neutral  alcohol,  and  dried  in  a  vacuum  at  105°  for  five 
hours.  After  cooling  in  a  dessicator,  the  samples  were  allowed  to  re- 
absorb moisture  from  the  atmosphere  for  several  days,  and  bottled. 
This  prepared  starch  could  then  be  weighed  out  in  the  open  balance 
without  errors  due  to  rapid  changes  in  weight  by  moisture  absorption. 
Its  moisture  content  was  determined.  These  starches  showed  no  dias- 
tatic activity  nor  reducing  sugars. 

Samples  of  this  air  dried  starch  were  weighed  into  250  cc  beakers, 
and  50  cc  of  water  added  to  each  at  27°  centrigrade.  Ten  cubic  centi- 
meters of  1%  dextrose  were  then  pipetted  in,  well  stirred,  and  allowed 
to  remain  at  27°.  At  the  end  of  exactly  one  hour  they  were  diluted  to 
100  cc  in  a  calibrated  volumetric  flask,  centrifuged  clear,  and  50  cc  of 
this  clear  supernatant  liquid  taken  for  determination  of  reducing 
sugars.  To  determine  whether  the  change  of  acidity  by  the  NagWO^ 
clarification  procedure  would  affect  the  adsorption  of  sugars  by  starch, 
and  to  check  up  on  the  effect  of  clarification  upon  Fehling's  solution 
reduction  (table  VIII,  page  33),  alternate  samples  were  clarified  at  the 
beginning  of  the  digestions.     In  other  words  3.  cc  of  NaaWO^  (15%) 

40 


and  .4  cc  cone.  HoSO^  were  added  immediately  after  the  10  cc  of  dex- 
trose solution.     The  results  are  shown  in  table  XI. 

TABLE  XI. 

The  recovery  of  reducing  sugars  after  addition  to  starch  in  water. 


^Uftcii  CI 

After  Correction 

umber 

Starch 

Sugars 

For  Volume 

as  Substrate 

pH 

Added 

By  A 

nalysis 

Displacement 

Grams 

MilHgranis 
Dextrose 

Mill 

i  grams 

Milligrams 

1. 

3.0 

5.092 

48.4 

50.4 

49.2 

2. 

3.0 

1.790 

48.4 

49.9 

48.8 

3. 

4.0 

5.000 

24.2 

25.2 

24.5 

4. 

4.0 

1.618 

24.2 

24.7 

24.0 

S. 

5.0 

5.581 

48.4 

50.4 

48.5 

6. 

5.0 

1.932 

48.4 

50.5 

48.7 

7. 

7.0 

5.466 

48.4 

53.5 

50.7 

8. 

7.0 

1.900 

48.4 
Maltose 

51.5 

48.6 

9. 

4.0 

4.600 

40.9 

43.8 

42.4 

10. 

4.0 

1.615 

40.9 

42.8 

41.5 

11. 

4.0 

1.900 

61.4 

64.9 

63.0 

The  last  column  of  figures  in  table  XI  shows  the  calculated  weights 
of  CugO  after  correcting  for  the  concentration  displacement  by  the 
starch  in  100  cc  volume.  The  corrected  values  fall  almost  within  the 
limit  of  experimental  error  with  the  exception  of  samples  numbers  1, 
7,  9  and  11.  The  good  agreement  of  the  pairs  of  results  also  confirms 
a  conclusion  of  table  VIII,  namely,  that  the  NagWO^  clarification  has 
no  efifect  on  Fehling  solution  reduction.  It  is  also  evident  from  the 
data  given  in  Table  XI  that  there  is  no  adsorption  of  reducing  sugars 
by  the  starch. 

Inhibition  of  Enzyme  Activity.  Much  has  been  published  on  the 
efifect  of  the  so-called  catalytic  poisons  on  various  enzymes.  The  re- 
sults are  so  numerous  and  the  conclusions  so  contradictory  that  it 
would  serve  no  purpose  to  review^  them  at  length.  It  has  been  shown 
that  each  enzyme  poison,  whether  it  be  an  antiseptic,  or  heavy  metal 
salt,  must  be  conisdered  separately  and  in  its  relation  to  the  particular 
enzyme  in  question.  In  general,  the  salts  of  the  heavy  metals,  antisep- 
tics, and  in  fact  most  of  the  so-cjffled  catalytic  poisons,  have  but  little 
inhibiting  efifect  on  the  activity  of  diastase  under  normal  conditions 
of  pH  and  salt  concentration  except  as  they  are  able  to  precipitate  the 
enzymic  carrying  protein  or  to  influence  the  pH  of  the  medium.  The 
work  of  Sherman  and  Caldwell  (1921),  has  further  emphasized  the 
protective  action  of  the  amino  acids  on  the  enzymes  in  the  presence  of 
catalytic  poisons.  Many  of  those  who  have  investigated  diastatic 
activity  have  recorded  the  use  of  NaOH  in  varying  concentrations  to 
stop  enzymic  action.     Others  have  employed  low  temperatures.     The 

41 


effect  of  continually  increasing  amounts  of  alkali  on  diastatic  diges- 
tions is  shown  later  in  Table  XVI  and  Fig.  3.  The  concentration  of 
alkali  required  for  complete  inhibition  of  flour  diastase  is  considerably 
higher  than  that  generally  recomended,  and  there  Would  seem  to  be 
serious  danger  of  destroying  some  of  the  sugars  present  by  the  addi- 
tion of  such  hydroxyl  ion  concentration,  as  pointed  out  by  Neff, 
(Armstrong,  1919.).  Furthermore,  the  increased  dispersion  and  solu- 
tion of  proteins  by  the  added  NaOH  is  not  desirable.  The  unsatisfac- 
tory results  obtained  in  preliminary  experiments,  whenever  NaOH 
was  used  to  stop  diastatic  activity,  or  lead  acetate  for  clarification  of 
flour  suspensions,  in  contradistinction  to  the  uniformly  satisfactory 
results  by  the  use  of  sodium  tungstate  and  sulphuric  acid,  lead  to  the 
experiments  which  are  summarized  in  Table  XII. 

Ten  grams  of  flour  were  weighed  out  into  300  cc  flasks  and  100  cc 
of  distilled  water  added  by  pipette,  with  continual  shaking  to  get  the 
flour  thoroughly  stirred  throughout  the  liquid.  The  various  mater- 
ials to  be  used  as  inhibiting  agents  were  then  added  and  the  whole 
allowed  to  remain  in  the  water  bath  for  the  lengths  of  time  as  desig- 
nated in  the  table,  (shaking  up  at  fifteen  minute  intervals).  Sample 
number  1  was  clarified  by  the  tungstate  reagent  at  the  end  of  the  first 
hour.  Sample  number  2  was  clarified  at  once,  then  allowed  to  stand 
one  hour  before  determining  the  reducing  sugars.  Sample  number  3 
was  clarified  in  the  same  manner  as  number  tw^o,  except  that  it  was 
allowed  to  stand  two  hours  before  determining  the  reducing  sugar.^. 
Sample  four  was  tried  with  only  3.  cc  of  sodium  tungstate,  onjtting 
the  precipitation  by  sulfuric  acid  until  the  end  of  the  hour.  Samples 
five  and  six  show  the  effect  of  two  different  concentrations  of  alkali 
previously  recommended  in  the  literature  for  the  inhibition  of  dias- 
tatic activity.  Samples  seven  and  eight  v/eve  treated  with  2.  cc  of 
basic,  and  2.  cc  of  neutral  lead  acetate,  respectively,  and  allowed  to 
stand  one  hour  before  filtration  and  removal  of  the  lead  for  sugar  de- 
terminations. They  were  then  clarified  and  treated  accord^'ng  to  the 
official  A.  O.  A.  C.  procedure.  Sample  number  7  was  cloud}^  and  re- 
quired twelve  hours  for  filtration,  while  sample  eight  filtered  much 
more  rapidly.  Samples  nine  and  ten  were  cooled  to  zero  degrees  by 
an  ice  and  salt  bath.  In  samples  1,  5,  6,  9,  and  10,  the  diastatic  activ- 
ities were  stopped  by  the  sodium  tungstate  clarification  in  the  manner 
described  above,  while  sample  4  required  the  addition  of  .4  cc  concen- 
trated H2SO4.  After  clarification  the  samples  were  all  made  up  to  a 
volume  of  200  cc,  centrifuged,  and  50  cc  aliquots  taken  for  reducing 
sugar  determinations.  The  results  are  expressed  in  table  XII  in  terms 
of  cuprous  oxide  corresponding  to  the  reducing  sugars  from  50.  cc  of 
the  clarified  solution  (2.5  grams  flour.) 

42 


TABLE  XII. 
The  effect  of  different  inhibiting  agents  on  the  diastase  of  flour. 

Weight   CU2O 
per  50.  cc's 


Sample  No. 

Inhibiting 
Agent 

Time  and 
Temperature 

of  Solution 
(2.5  g.  flour) 

pHof 
Digestion 

1. 

none 

1  hr.  at  27° 

.0833 



2. 

(Na^WO* 
+  H.SO4) 

1  hr.  at  27° 

.0140 

below  2.0 

3. 

(Na.WO* 
+  H,SO0 

2  hrs.  at  27° 

.0141 

below  2.0 

4. 

(Na.WO* 
only) 

1  hr.  at  27° 

.0491 

7.241 

5. 

2.  ccO.lN. 
NaOH) 

1  hr.  at  27° 

.0316 

6. 

(5cc  O.IN. 
(NaOH) 

1  hr.  at  27° 

.0169 

9.1336 

7. 

PbAc  (Neut.) 

1  hr.  at  27° 

.1034 

8. 

PbAc  (Basic) 

1  hr.  at  27° 

.0066  • 

9. 

Ice 

1  hr.  at  0° 

.0287 

5.981  (21.°C) 

-      10. 

Ice 

3  hrs.  at  0° 

.0388 

5.981  (21.°C) 

Confirmatory  results  similar  to  these  and  leading  to  the  same  con- 
clusions have  been  obtained  on  two  other  flours  (Our  No.  1  and  1003). 

Samples  numbers  2  and  3  of  Table  XII,  as  examples  typical  of  many 
similar  determinations,  show  conclusively  that  the  use  of  sodium  tung- 
state  and  sulfuric  acid,  with  a  final  pH  of  between  1.5  and  2,  effectual- 
ly stop  diastatic  activity.  This  is  accomplished  both  by  complete  pre- 
cipitation of  the  enzymic  active  protein  and  by  effecting  a  hydrogen- 
ion  concentration  which  itself  Inhibits  diastatic  activity.  The  concen- 
tration of  acid  is  not  sufficient  to  further  hydrolyse  the  disacharides  or 
dextrins,  even  after  several  hours  standing.  This  had  been  previously 
pointed  out  by  Swanson  and  Calf  in,  who  used  a  final  concentration  of 
.02N  H2SO4,  which  corresponds  to  a  pH  of  somewhere  between  2  and 
2.5.  This  fact  allows  an  accurate  measurement  of  the  reducing  sug- 
ars by  gravimetric  Fehling's  determination  at  the  operator's  conven- 
ience. 

Samples  5  and  6,  Table  XII,  indicate  that  NaOH  is  not  a  reliable 
inhibiting  agent  to  use  for  diastatic  preparations  where  the  quantity 
of  buffer  is  as  high  as  that  found  in  flours  or  malts,  unless  a  large  con- 
centration is  used.     There  is   also  the   probable  ganger   of   destroying 

43 


part  of  the  reducing  sugars  present  if  too  high  a  concentration  of  al- 
kali be  used. 

The  use  of  lead  acetate,  (table  XII,  numbers  7  and  8),  gives  variable 
results  depending  upon  the  concentration  of  the  reagent,  the  concen- 
tration of  the  material  used  to  de-lead  the  solution,  and  the  time  con- 
sumed in  the  operations  of  filtration. 

In  point  of  time  alone  the  sodium  tungstate  procedure  requires  only 
about  five  minutes,  with  the  elimination  of  all  filtration,  which  is  a 
great  advantage  in  enzyme  work,  while  the  clarified  solution  can  be 
allowed  to  stand  for  several  hours,  or  until  all  of  the  samples  are 
ready  for  the  Fehling  reduction. 

Samples  numbers  9  and  10  (table  XII),  clearly  indicate  that  the 
temperature  of  an  ice  bath  will  not  stop  diastatic  activity  in  solutions 
where  the  diastase  is  protected  by  buffers  naturally  occurring  in  the 
plant  medium.  In  the  two  trials  the  temperature  within  the  flasks 
was  held  at  zero  by  means  of  salt  and  ice,  and  they  had  to  be  stirred  at 
short  intervals  to  prevent  ice  crystals  forming  on  the  inside  surface  of 
the  flasks.  Other  samples  with  temperatures  around  5°C  showed  very 
nearly  the  same  results.  Samples  2  and  3  (Table  XII),  in  conjunction 
with  results  of  nearly  a  hundred  other  similar  determinations,  serve  to 
show  this  "blank"  gives  very  constant  and  duplicable  results,  and  af- 
fords a  measure  of  the  reducing  sugars  present  in  the  flour.  While 
this  reduction  of  Fehling  solution  is  due  to  dextrose,  and  the  total 
reduction  by  autolytic  digestion  of  the  flour-water  suspension  is  a  re- 
sult of  added  maltose  produced  by  diastatic  action,  the  difference  in 
the  weight  of  CugO  produced  give  a  reliable  measure  of  the  maltose. 
This  procedure  for  the  determination  of  original  reducing  sugars  in 
the  sample  should  be  directly  comparable  to  the  values  obtained  from 
the  official  alcohol  extraction  method.  Likewise,  it  should  be  pos- 
sible to  substitute  the  sodium  tungstate  clarification  for  the  lead 
acetate  procedure  in  the  alcohol-extract  of  sugars  from  flour  and  malt 
products.  Preliminary  experiments  on  the  application  of  this  new 
procedure  for  both  reducing  and  total  sugars  have  indicated  its  suc- 
cessful application.  Collaborative  work  with  the  Department  of  Ag- 
riculture, Bureau  of  Chemistry,  is  now  being  conducted  on  this  prob- 
lem and  the  results  will  be  reported  at  a  later  date. 

Effect  of  Concentration.  In  general,  investigators  of  enzymic  ac- 
tivities have  found  it  necessary  to  take  the  concentration  effect  into 
consideration  before  establishing  their  conditions  for  observations  and 
measurements  on  enzyme  preparations.  This  concentration  effect  is 
especially  important  when  working  with  extracted  and  purified  en- 
zyme preparations.  In  the  preliminary  discussion  of  factors  which 
may  limit  the  activity  of  diastase  in  one  way  or  another  two  such  fac- 

M 


tors  were  considered,  namely,  amyloclastic  power,  and  variable  resist- 
ance of  different  starches  to  amyloclastic  action. 

As  a  third  limiting  factor  the  concentration  effect  on  diastatic  ac- 
tion should  receive  attention  because  of  its  peculiar  relation  in  this 
series  of  measurements.  Before  diastatic  enzymes  can  act  to  best  ad- 
vantage there  must  be  sufficient  water  present.  In  the  germinating 
seed  this  is  assured  and  controlled  through  imbibition  of  water  by  the 
colloidal  system.  In  a  dough  batch  the  concentration  is  likewise  con- 
trolled by  the  amount  of  water  the  flour  colloids  can  imbibe  and  still 
retain  sufficient  elasticity  and  tenacity  to  handle  w'ell  in  baking  prac- 
tice. (Ostwald  1919,  and  Luers  and  Ostwald  1919-1921).  This  de- 
gree of  concentration  varies  considerably  with  different  flours  and  in 
the  hands  of  different  bakers,  within  certain  limits.  When  compared 
to  the  long  period  of  time  required  for  germination,  the  short  fermen- 
tation of  a  dough  should  show  concentrations  which  are  more  or  less 
comparable. 

To  determine  what  effect  concentration  wK)uld  have  on  the  activity 
of  diastase  as  measured  by  the  methods  employed  in  this  report,  two 
types  of  experiments  were  carried  out.  In  one,  attempts  were  made 
to  measure  the  reducing  sugars  produced  in  a  dough  fermentation 
containing  percentages  of  water  comparable  to  the  normal  absorption 
as  obtaining  in  bake  shop  practice,  and  in  the  other,  to  determine  the 
concentration  effect  in  dilutions  such  as  are  used  in  the  method  fi- 
nally chosen  for  measurements  of  comparative  diastatic  powers  in  the 
standard  flour  samples. 

In  Table  XIII  the  first  three  values  given  are  those  for  diastatic 
activity  in  the  doughs,  and  were  obtained  as  follows :  50  grams  of 
flour  were  weighed  into  250  cc  beakers  and  allowed  to  come  to  a  tem- 
perature of  27°C.  Distilled  water  at  27°C  was  added  from  a  burette 
in  volumes  ranging  from  27.5  cc  to  50  cc,  or  in  other  words  in  amounts 
corresponding  to  absorptions  of  55  to  100%.  The  flour  and  water  was 
thoroughly  doughed  up  by  means  of  a  stiff  spatula  and  the  stiffer 
doughs  were  kneaded  a  minute  in  the  fingers.  This  dough  was 
pressed  into  the  bottom  of  the  beaker,  covered,  and  placed  in  the  water 
bath  of  27°  for  one  hour.  At  •fhe  end  of  that  time  the  dough  was 
weighed  and  one-fifth  of  the  total  weight,  corresponding  to  ten  grams 
of  flour,  was  taken  for  determination  of  sugars.  This  aliquot  sam- 
ple of  dough  was  transferred  to  a  250  cc  bottle,  100  cc  of  water 
were  added,  and  several  drops  of  50%  NaOH  to  aid  in  dispersing  the 
gluten  and  slow  up  enzymatic  activity.  The  bottle  was  quickly  stop- 
pered and  shaken  for  a  few  minutes  to  completely  disperse  the  gluten 
and  dissolve  out  the  sugars.  The  contents  of  the  bottle  were  then 
rinsed  into  a  200  cc  volumetric  flask  and  clarified  by  sodium  tungstatc 

45 


and  sulfuric  acid  as  described  above.  From  the  centrifuged  super- 
natant solution  50  cc  aliquots  were  pipetted  out  for  reducing  sugar 
determinations.  The  values  for  the  other  four  concentrations  as  re- 
corded in  the  same  table  were  obtained  by  varying  the  ratios  of  the 
flour-water  suspensions  from  1  :  2.5  up  to  1  :  40.  and  allowing  the  mix- 
ture to  digest  autolytically  for  one  hour  at  27°.  The  diastase  in 
these  samples  was  then  stopped  by  the  regular  sodium  tungstate  pro- 
cedure, made  up  to  volume,  and  the  reducing  sugars  determined  in 
the  centrifugate  in  the  same  manner  as  for  the  other  three  samples. 

TABLE  XIII. 

The  effect  of  varying  ratios  of  flour  to  water  on  the  diastatic 

activity  of  a  wheat  flour. 


Ratio 
1:0.55 


Weight  of 

H.O 

Sample 

Flour 

Volumes 

Number 

Grams 

cc 

3. 


6. 


50. 


50. 


50. 


10. 


10. 


10. 


10. 


27.5 


35.0 


50.0 


25.0 


75.0 


100.0 


400.0 


1:0.70 


1:1.0 


1:2.5 


1:7.5 


1:10.0 


:40.0 


Weighed 

as  CU2O 

per  2.5 

Grams  FlourlO, 

Grams 

Calculated 

Weight  of 

CU2O  per 

. '  Gram  Flour 

Grams 

.0675 
.0665 

.2680 

.0727 
.0723 

.2900 

.0862 
.0853 

.3430 

.0835 
.0835 
.0827 

.3348 

.0838 
.0831 
.0836 

.3340 

.0833 
.0836 
.0830 
.0839 

.3338 

.0835 
.0835 
.0820 

.3340 

The  low  results  for  samples  numbers  1  and  2,  table  XIII,  are  prob- 
ably due  to  an  insufficiency  of  water  to  allow  complete  enzymatic  ac- 
tivity. The  normal  dough  for  this  flour  would  require  29.5  cc  of  water 
for  50  grams  of  flour,  which  is  less  than  the  amount  added  to  sample 
number  two.  It  must  be  understood,  therefore,  that  diastatic  action 
is  not  complete  in  a  normal  dough  under  average  bake  shop  conditions. 
The  result  for  diastatic  activity  in  sample  number  three  on  the  other 
hand,  is  too  high  by  about  3.  percent,  yet  it  falls  within  the  range  of 
values  as  determined  by  the  other    samples.     Considering,    however, 


46 


the  necessary  inaccuracies  of  method  in  controlling  temperatures,  in 
doughing  up  the  flour  samples,  in  weighing  and  dividing  the  dough, 
and  especially  in  the  time  required  to  disperse  the  dough  and  dissolve 
out  the  sugars  before  the  enzymic  activity  can  be  inhibited  by  clari- 
fication, the  agreement  with  the  samples  numbers  4  to  7  is  about  as 
close  as  could  be  expected.  Attention  might  be  called  to  the  fact  that 
the  results  as  expressed  in  column  five  contain  the  errors  of  the  actual 
determinations  multiplied  four  times. 

The  excellent  agreement  of  the  results  for  autolytic  digestion  of 
the  flour  with  water  in  ratios  of  1 :1  or  more,  over  so  wide  a  range  of 
concentrations,  eliminates  one  of  the  most  troublesome  factors  in  a 
determination  of  this  nature.  It  enables  one  to  add  other  reagents  or 
to  vary  the  total  valume  of  the  solution  at  will  and  yet  have  only  the 
effect  of  the  one  desired  variable  imposed  on  the  results. 

It  is  evident  here  that  the  enzyme  and  its  substrate  exhibit  an  in- 
timate relationship.  The  large  and  practically  constant  excess  of 
starch  substrate  is  available  for  the  relatively  small  amount  of  en- 
zyme which  exists  within  the  plant  material  constituting  the  flour 
particles  and  which  is  held  in  close  contact  with  the  starch  granules 
on  which  it  must  act.  When  sufficient  water  has  been  added  to  prop- 
erly hydrate  the  bio-colloids  and  enable  the  enzymes  to  function,  any 
further  increase  in  the  amount  of  water  has  but  little  effect,  the 
enzymes  having  been  adsorbed  to  the  substrate  and  not  immediately 
subject  to  dilution.  This  is  not  the  case  with  the  solutions  of  "puri- 
fied" and  dissolved  diastatic  preparations  or  extracts,  which  do  show 
some  concentration  effect  because  of  their  suspension  throughout  the 
solution.  The  results  of  Thatcher  and  Koch  described  above,  as  well 
as  the  work  of  nearly  all  of  the  other  investigators  of  diastase,  have 
shown  that  a  part,  never  all,  of  the  diastatic  activity  could  be  extracted 
from  flour  as  a  solution.  But  these  extracts  do  not  show  normal,  i.  e. 
natural,  diastasis  as  in  the  plant  tissue.  Frequent  shaking  and  pro- 
longed digestion  are  necessary  for  such  a  removal  of  enzymes.  It 
should  be  stated  here  in  this  connection  that  too  frequent  and  violent 
shaking  of  the  flour-water  suspensions  used  in  this  work  decreased 
considerably  the  quantities  of  maltose  produced  per  unit  of  time,  and 
the  supernatant  liquid  evidenced  increasing  diastatic  power.  It  was 
for  that  reason  that  the  suspensions  vi^ere  agitated  only  at  fifteen 
minute  intervals  by  rotating  the  containing  flasks. 

But  by  far  the  more  important  contribution  of  this  set  of  results  to 
the  problem  in  hand  rests  on  the  demonstration  that  autolysis  of  flour- 
water  suspensions  in  ratios  around  1 :10  as  carried  out  in  this  series  of 
experiments  can  be  consideried  as  furnishing  a  logical  laboratory  basis 
for  the  measurement  of  diastatic  capacity  of  the  flour  when  mixed  in  a 

47 


dough.  The  temperature  is  taken  as  the  average  accepted  fermenting 
temperature  for  ordinary  bake  shop  practice.  The  time  may  be  con- 
trolled at  will.  The  pH  of  the  digestion  mixture  is  controlled  by  the 
flour,  usually  between  5.7  and  6.1,  and  is  close  to  that  at  which  the 
dough  fermentation  normally  begins.  The  acidity  which  develops  as 
fermentation  proceeds  in  the  dough  gradually  increases  to  a  pH  value 
between  4.8  and  5.4,  at  which  point  the  diastatic  activity  is  at  or  very 
near  its  maximum.  Sherman  and  Thomas  (1915),  and  Sherman, 
Thomas,  and  Baldwin  (1919),  showed  that  the  protective  action  and 
stabilizing  effect  of  buffer  salts  on  diastase  is  very  marked  both  with 
respect  to  concentration  and  temperature.  This  buffer  effect  undoubt- 
edly accounts  in  part  for  the  stability  of  the  enzyme  rate  by  the  meth- 
od of  measurement  employed  here.  This  is  also  shown  further  on  by 
the  data  on  relation  of  pH  to  diastatic  activity. 

The  relations  between  autolytic  measurements  and  diastatsis  in  the 
dough  during  a  normal  fermentation  are  discussed  further  in  connec- 
tion with  measurements  of  maltose  production  in  doughs. 

It  would  seem  advantageous  then  from  the  standpoint  of  sugar 
production  to  mix  doughs  with  the  maximum  amount  of  water  which 
they  will  carry  and  still  work  well.  The  machinery  designed  for  the 
modern  large  baking  plants  wnll  handle  doughs  with  absorption  as 
high  as  70%,  which  allows  practically  maximum  maltose  production 
by  diastatic  action. 

Method  for  Measuring  Diastatic  Power  of  Flour.  As  a  result  of  the 
data  obtained  up  to  this  point  a  definite  method  was  developed  for  the 
determination  of  diastatic  power  in  flour.  This  procedure  was  used 
with  variations  in  time,  temperature,  or  pH,  as  the  case  might  require, 
for  all  the  experiments  which  follow.  10.  gram  samples  of  flour  are 
weighed  out  and  transferred  to  250  or  300  cc  Erlenmeyer  flasks.  These 
are  placed  in  a  water  thermostat  and  brought  to  a  temperature  of  ex- 
actly 27° C.  A  flask  containing  a  sufficient  volume  of  distilled  water 
is  also  placed  in  the  bath  and  kept  at  27° C  for  subsequent  use.  By 
means  of  a  pipette  100.  cc  of  the  distilled  water  are  run  into  the  sam- 
ple, while  rapidly  rotating  the  flask  to  obtain  a  thorough  suspension 
of  the  flour.  The  last  few  cc's  in  the  pipette  are  allowed  to  rinse  the 
material  down  from  the  sides  of  the  flask.  The  flask  is  quickly  re- 
placed in  the  thermostat,  stoppered  loosely,  and  allowed  to  remain 
exactly  60  minutes.  A  few  minutes  after  starting  the  digestion  the 
flask  is  rotated  to  stir  up  the  suspension  and  hasten  the  equalization 
of  temperatures,  and  the  shaking  is  repeated  at  fifteen  minute  inter- 
vals. At  the  end  of  the  digestion  period  the  contents  of  the  flask 
are  quickly  rinsed  into  a  200  cc  volumetric  flask,  diluted  to  about  175. 
cc  and  clarified. 

48 


To  clarify,  first  make  sure  that  the  solution  is  neutral  or  slightly 
alkaline.  Five  drops  of  0.04%  Thymol  Blue  serves  as  a  convenient 
indicator,  appearing  cream-yellow  in  color  when  neutral,  or  blue  when 
slightly  alkaline.  Add  3  cc  of  a  15%  solution  of  sodium  tungstate 
(Na2W04.2H20)and  mix  thoroughly  with  the  flour  suspension.  Then 
add,  drop  by  drop,  from  a  1  milliliter  graduated  pipette,  with  con- 
stant shaking,  sufficient  concentrated  HgSO^  to  turn  the  indicator  a 
decided  pink  color,  with  two  or  three  drops  in  excess.  Four-tenths 
of  a  milliliter  (0.4  cc)  are  usually  sufficient  if  the  original  flour  suspen- 
sion was  nearly  neutral.  This  clarification  likewise  serves  to  stop 
the  enzymatic  activity  and  prevents  further  change  in  sugar  content. 
Dilute  to  the  mark,  shake  thoroughly,  pour  into  centrifuge  cups,  and 
whirl  for  about  five  minutes.  By  means  of  a  calibrated  pipette  trans- 
fer 50.  cc  of  the  clear  supernatant  liquid  to  a  400.  cc  pyrex  beaker 
for  the  determination  of  reducing  sugars  by  the  Munson-Walker  offi- 
cial method. 

A  blank  is  also  run  at  the  same  time  as  the  sample  to  correct  for  the 
natural  reducing  sugars  in  the  flour.  It  likewise  gives  a  measure  of 
these  reducing  sugars.  To  prepare  the  blank,  mix  100.  cc  of  Water  at 
27°C  and  10.  grams  of  flour  and  immediately  inhibit  diastatic  activity 
by  clarifying  with  the  sodium  tungstate  in  the  manner  just  described. 
The  blank  determination  is  then  carried  out  in  the  same  manner  as  the 
samples  except  that  the  addition  of  0.4  cc  of  concentrated  H2SO4  is 
omitted  on  dilution  to  volume.  It  does,  no  harm  to  allow  the  clari- 
fied solution  in  the  beakers  to  stand  an  hour  or  two  until  other  samples 
are  ready.  When  making  the  Fehling  reduction  the  excess  acidity 
of  the  solution  can  conveniently  be  neutralized  by  using  a  predeter- 
mined number  of  drops  of  strong  NaOH.  Following  the  reduction 
of  the  Fehling  solution  the  CU2O  is  filtered,  washed,  dried,  and 
weighed  in  the  gooch  crucibles.  Subtract  the  weight  of  the  CugO 
corresponding  to  the  blank  from  that  of  the  sample.  The  result  is  the 
CugO  corresponding  to  the  maltose  produced  by  the  diastase  in  2.5 
grams  of  flour.  This  value  for  anhydrous  maltose  as  found  from  the 
tables,  multiplied  by  4.  gives  the  diastatic  power  per  10.  grams  of 
flour.  This  procedure  has  been  fotfnd  by  many  repeated  trials  on  dif- 
ferent flours  to  give  accurate  results  which  could  be  duplicated  with- 
out difficulty. 

The  temperature  of  27°C  is  chosen  because  that  is  very  near  the 
average  of  the  temperatures  at  which  doughs  are  fermented  in  com- 
mercial practice.  The  proofing  temperature,  usually  around 
32.5°C  is  varied  widely  in  practice,  and  continues  at  that  temperature 
but  a  short  time  compared  to  the  total  fermentation  time.  Ten  grams 
are  chosen  for  a  sample  because  the  reducing  sugars  produced  give  a 

49 


convenient  weight  of  CugO  on  the  asbestos  mat.  The  200  cc  final 
volume  is  selected  because  it  conveniently  fills  tv^o  100.  cc  centrifuge 
tubes  v^hich  balance  each  other.  How^ever,  no  error  is  introduced  by 
a  change  of  final  volume  to  a  250,  300  cc  or  500  cc  flask,  and  the  latter 
is  sometimes  desirable  v^hen  working  with  a  sample  of  unusually  high 
diastatic  power,  such  as  a  malt  flour. 

The  application  of  this  procedure  to  a  determination  of  malt  prep- 
arations for  use  in  panary  fermentation  require  some  special  consider- 
ations, and  will  be  left  for  discussion  further  on  in  this  paper. 

Effect  of  Time  and  Temperature  on  Activity  of  Flour  Diastase.  4. 
Temperature.  The  factors  of  greatest  importance  in  the  limitation 
of  enzymatic  activity  have  been  shown  by  many  investigators  to 
be  those  of  temperature,  hydrogen  or  hydroxyl-ion  concentration, 
and  of  time.  Of  these,  the  temperature  factor  will  be  considered  first 
because  it  is  by  far  the  most  significant  in  determining  the  produc- 
tion of  maltose  by  the  action  of  diastase  under  the  conditions  obtain- 
ing in  a  bread  dough.  Because  of  the  very  high  rate  of  autolytic  dias- 
tasis in  these  flour-water  suspensions  at  temperatures  near  the  range 
of  optimum  temperatures  it  was  difficult  to  prevent  large  errors  in 
the  results  between  50.°  and  70.°C.  Slight  variations  in  temperature 
for  the  first  few  minutes  of  digestion  vitiated  the  value  of  the  deter- 
mination. Preliminary  experiments  in  connection  with  temperature 
control  had  shown  that  a  few  minutes  heating  of  the  dry  flour  in  flasks 
in  the  water  bath  had  no  effect  on  the  diastatic  activity  providing  the 
temperature  was  not  above  65° C.  Prolonged  heating,  however,  pro- 
duces a  very  slow  reaction  due  no  doubt  to  the  partial  vaporization  of 
the  moisture  in  the  flour  and  its  condensation  on  the  sides  of  the  flask 
with  consequent  wetting  of  some  of  the  flour.  After  an  hour  of  heat- 
ing at  70°C  a  sample  of  flour  was  found  to  increase  slightly  in  reduc- 
ing sugars  and  to  decrease  in  diastatic  powxr. 

The  procedure  followed  in  this  series  was  to  first  bring  the  water 
thermostat  up  to  the  temperature  desired.  The  10.  gram  samples  of 
flour,  in  loosely  stoppered  erlenmeyer  flasks,  were  placed  in  this  bath 
about  ten  minutes  before  the  water  was  added.  Exactly  100.  cc  of 
distilled  water  in  150  cc.  erlenmeyer  flasks,  were  heated  to  the  desired 
temperature  in  the  water  bath  and  poured  quickly  on  to  the  flour 
sample.  A  sensitve  100° C  thermometer  was  placed  in  the  flask  before 
adding  the  water.  The  temperatures  resulting  from  the  mixing  of 
flour  and  water  were  usually  a  few  tenths  of  a  degree  below  that  de- 
sired. They  were  quickly  corrected  by  rapidly  rotating  the  flask  over 
the  hot  spot  of  a  wire  gauze  heated  by  a  bunsen  flame,  and  the  flask 
then  replaced  in  the  water  bath.  At  the  end  of  the  desired  time  the 
flask  was  removed  from'  the  bath,  transferred  to  a  200  cc  volumetric 

SO 


flask,  clarified,  cooled  when  necessary,  diluted  to  volume,  centrifuged, 
and  the  reducing  sugars  determined  as  described  above.  Figure  1 
shows  graphically  the  nearly  autocatalytic  nature  of  the  temperature 
curve  for  one  hour  digestion.  Three  hour  digestions  gave  a  curve  of 
the  same  kind.  The  values  from  which  the  curves  are  plotted  are  ex- 
pressed in  Table  XIV  as  grams  of  maltose  produced  by  the  autolytic 
diastasis  in  10.  grams  of  flour.  These  values  are  obtained  from  the 
weights  of  CugO  corresponding  to  the  50.  cc  aliquots  of  the  digestion 
solution.  The  blank  determination,  representing  the  natural  reducing 
value  of  the  extracted  flour-sugars,  in  terms  of  Cu^O,  was  subtrated 
from  the  total  weight  of  CU2O  as  obtained  from  the  auto-diastasis. 
The  difference  gave  the  cuprous  oxide  equivalent  to  the  maltose  pro- 
duced by  diastasis  alone.  The  corresponding  weight  of  maltose  found 
from  the  Munson-Walker  table  was  multiplied  by  the  necessary 
aliquot  number  to  obtain  the  value  per  ten  grams  (total)  of  sample. 
Each  value  is  the  average  of  at  least  three  reducing  sugar  determina- 
tions for  that  time  and  temperature.  The  maltose  values  are  calcu- 
lated as  *'diastatic"  maltose,  in  grams. 


TABLE  XIV. 

The  relation  of  temperature  to  the  activity  of  wheat  flour  diastase. 

10.  grams  flour  No.  1009  used  for  each  sample. 


Temperature 

Degrees 

Centigrade 

0. 
27. 
25. 
55. 
60. 
63.5 
65. 
67. 
70. 

75-76 
♦82-83 


Weight  of  Maltose 

from  Diastasis  of 

10.  grams  Flour. 

1  Hour  Digestion 

Gram$ 

.0386 

.2118 

.3238 
1.1378 
2.1396 
2.9067 
1.6408 

.7698 

.4016 

.0922 

.0244 


♦Gelatinized. 


The  maximum  production  of  maltose  was  29%  at  63.5°C.  But  this 
temperature  ( 146.3°  F)  is  probably  never  reached  in  a  dough  during 
a  normal  fermentation,  and  only  for  a  few  minutes  while  baking. 


51 


.  20  30 

Temperature 


FIGURE  1. 
The  effect  of  temperature  on  the  activity  of  wheat  flour  diastase. 

5.  Time.  The  relationships  between  activity,  temperature,  and 
time,  can  be  conveniently  considered  together.  Since  the  rate  of  dias- 
tasis is  so  enormously  increased  at  higher  temperatures,  the  curves  for 
the  production  of  maltose  with  time  must  show  a  corresponding  in- 
creased initial  rate  at  higher  temperatures.  Consequently  the  time 
curves  for  several  temperatures  were  determined  and  are  plotted  in 
Figure  2.  The  data  corresponding  to  these  curves  is  tabulated  in 
Table  XV  in  groups  of  results,  one  set  for  each  curve.  To  simplify 
tabulation  the  total  values  per  10.  gram  sample  are  calculated  from 
the  actual  weighings  as  described  for  the  values  in  Table  XIV.  The 
last  set  of  data  in  Table  XV  is  obtained  on  a  different  flour  (our 
laboratory  sample  No.  1)  and  is  also  included  in  Figure  2  to  show 
the  similarity  of  the  results  for  a  flour  of  lower  diastatic  power. 


TABLE  XV. 
The  variation  of  diastatic  activity  in  wheat  flour  with  time. 

Grams  of  Maltose  Produced  by  Diastase  in  10.  grams  of  Flour 
Time  in  Minutes 


Flour 

1 

uirtiber 

Temp.     30 

60 

120 

1009 

o.-c 

.0383 

1009 

27.'C 

.2114 

.2942 

1009 

35. "C.    .2334 

.3235 

.4497 

1009 

55.x.    .5608 

1.0380 

1.2464 

1009 

64-65.'C 

1.5402 

3.0796 

1009 

69-70.X 

.3801 

.8225 

1 

2i:C.        .0725 

.0963 

.1313 

180 

.0702 

.3578 

.5181 

1.3862 

2.6718 

!i423 


240 


.3950 

.5832 

1.4916 


300 

.4420 

1,4820 


,1632 


.1741 


360 

!4650 
.6230 


,1870 


r52 


60 

Time  -  minutes 


FIGURE  2. 

The  change  of  activity  of  wheat  flour  diastase  with  time, 
at  different  temperatures. 


The  curves  drawn  from  the  data  yn  Table  XV  resemble  very  closely 
those  which  Collatz  and  Bailey  obtained  for  the  increase  of  conductiv- 
ity in  flour  extracts  due  to  the  action  of  the  enzyme  phytase  on  the 
phytin.  It  would  appear  probable  that  these  two  enzymes  exhibit  a 
parallel  activity  for  a  given  flour  at  any  particular  temperature. 

The  rate  of  maltose  production  has  reached  practically  a  constant 
value,  for  ordinary  fermentation  temperatures,  between  two  and  three 
hours  diastasis.  In  the  case  of  Flour  No.  1,  Table  X\^,  the  graph  for 
maltose  production  with  time  is  nearly  a  straight  line  from  two  to 
twenty-four  hours. 

S3 


6.  Acidity.  The  diastases  of  many  biologically  different  materials 
of  both  plant  and  animal  origin,  have  been  investigated  and  reported 
in  the  literature.  The  temperature  optima  vary  considerably  for  dif- 
ferent samples,  no  doubt  due  to  adaptations  to  environment.  The 
agreement,  however,  of  the  optima  for  hydrogen  ion  concentrations 
of  most  of  the  diastases  reported  seems  to  point  to  a  general  relation- 
ship of  activity.  Because  of  the  close  relationship  between  the  barley 
and  wheat  grains  the  diastase  of  wheat  would  be  expected  to  show  an 
optimum  activity  at  a  pH  of  4.7  to  5.0,  corresponding  to  that  for  the 
diastase  of  malted  barley  reported  by  Sherman  and  Walker,  and  Sher- 
man, Thomas  and  Baldwin. 


TABLE  XVI. 

The  influence  of  hydrogen  ion  concentration  on  the  activity  of 
wheat  flour  diastase. 

10.  gram  samples.    Flour  No.  1009.    1.  hour  digestion  @  27  C. 


Weight  of 

Corre- 
sponding 

CuzO  in  Milligrams 

Weight  of 

Maltose  in 

pH  After 

Weight 

Weight 

Milligrams 

Acid( 

or  Alkali 

1  Hour 

per  50.  cc 

per  10  g. 

per  10  g. 

Added 

Di{?estion 

Aliquot 

Flour 

Flour 

25.     cc 

N/10  HCl 

1.946 

15.7 

62.8 

+  • 

10.     cc 

N/10  HCl 

2.775 

27.5 

110.0 

34.8 

15.     cc 

N/25  HCl 

3.522 

78.4 

313.6 

196.1 

10.     cc 

N/25  HCl 

4.006 

109.6 

438.4 

295.1 

10.     cc 

N/25  HCl 

4.034 

111.8 

447.2 

302.2 

7.5  cc 

N/25  HCl 

4.399 

115.4 

361.6 

313.5 

5.0  cc 

N/25  MCI 

4.808 

116.5 

465.9 

316.8 

3.0  cc 

N/25  HCl 

5.112 

111.3 

445.2 

300.6 

2.5  cc 

N/25  HCl 

5.195 

112.6 

450.4^ 

304.7 

0.0 

5.691 

83.7 

334.8\ 
335.4/ 

213.4 

0.0 

5.742 

83.8 

2.5  cc 

N/25  NaOH 

6.352 

45.1 

180.4 

90.7 

5.0  cc 

N/25  NaOH 

7.023 

34.7 

138.8 

57.4 

7.5  cc 

N/25  NaOH 

7.527 

25.2 

100.8 

27.4 

10.0  cc 

N/25  NaOH 

9.067 

19.5 

78.0 

9.9-f.: 

5.0  cc 

N/25  NaOH 

9.134 

16.5 

66.0 

+.  - 

6.0  cc 

N/10  NaOH 

9.968 

16.0 

64.0 

+. 

Blank 



14.1 

56.4 

Figure  3  is  a  graphical  representation  of  the  data  in  Table  XVI 
and  shows  the  production  of  maltose  by  diastatic  enzymes  with  vary- 
ing concentrations  of  hydrogen  and  hydroxyl-ions,  in  terms  of  pH. 
To  obtain  the  data  from  which  Table  XVI  is  compiled,  pairs  of  sam- 
ples were  run  simultaneously.     The  regular     procedure     described 

54 


above  was  used  for  each  sample  in  which  the  reducing  sugars  were  to 
be  determined,  acid  or  alkali  being  added  as  shown  in  the  table  at  the 
beginning  of  the  digestion  to  produce  the  desired  pH.  The  other,  or 
check  sample  was  treated  in  exactly  the  same  manner  up  to  the  end 
of  the  digestion  period,  when  instead  of  clarifying,  the  suspension  was 
thoroughly  shaken  up,  poured  directly  into  centrifuge  cups,  centri- 
fuged,  and  the  supernatant  liquid  at  once  subjected  to  pH  measure- 
ments. In  this  manner  the  resulting  pH  at  the  end  of  the  one  hour 
digestion  was  measured  for  each  sample.  For  convenience  in  drawing 
the  curves  in  Figure  3,  the  weights  of  CugO  equivalent  to  the  diastatic 
activity  were  used  to  prepare  the  graph  (Figure  3),  rather  than  the 
corresponding  weights  of  maltose.  This  was  because  the  lower  mal- 
tose values  were  too  small  to  be  accurately  determined  from  the  mal- 
tose tables.  However,  the  results  are  expressed  in  milligrams  of  mal- 
tose actually  produced  by  the  diastase  in  ten  grams  of  flour.  The  cal- 
culation of  results  in  this  manner  have  been  described  above. 


FIGURE  3. 

The  relationship  between  the  activity  of  wheat  flour  diastase 
and  the  pH  of  the  medium. 


The  diastatic  activity  of  wheat  exhibits  practically  the  same  maxi- 
mum pH  as  that  found  for  barley  malt.  This  maximum  occurs  at  a 
pH  of  4.7  to  4.8,  with  a  broader  range  of  maxima  between  4.0  and  5.3. 


55 


The  rapid  decrease  in  the  production  of  maltose  in  the  ranges  of  pH 
from  5.  to  6.5  may  be  of  particular  significance  in  the  fermentation 
of  different  grades  of  flour.  It  may  also  account  for  some  of  the  dif- 
ferences in  baking  strength  which  become  apparent  with  aging  of  the 
flour. 

The  hydrogen-ion  concentration  of  the  normal  bread  dough  when 
mixed  is  very  approximately  that  of  the  flour,  usually  slightly  higher, 
and  in  the  case  of  the  flours  here  used  varies  from  a  pH  of  6.15  to  pH 
5.6.  The  optimum  pH  for  diastatic  activity  is  never  reached  in  the 
short  fermentation,  straight  dough  process,  for  a  normal  dough,  and 
not  often  in  sponge  doughs.  Even  in  the  latter  case  such  a  high  acid- 
ity is  obtained  only  after  six  or  more  hours  of  fermentation,  at  which 
time  the  diastase  has  suflfered  greatly  in  loss  of  activity  due  to  changes 
of  hydration  of  the  colloidal  proteins.  As  the  fermentation  advan- 
ces the  hydrogen-ion  concentration  slowly  increases,  until  an  appar- 
ent miximum  is  reached  somewhere  around  a  pH  5.4  to  5.2,  when  the 
dough  is  ready  for  the  oven.  This  point  may  well  be  that  condition 
in  which  the  COg  produced  by  the  zymase  of  the  yeast  cells  has  sat- 
urated the  dough.  The  effect  of  this  increase  of  acidity  may  then  be 
regarded  as  reciprocal,  first  the  increasing  hydrogen-ion  concentration 
from  a  pH  of  6.  to  pH  5.  has  the  efifect  of  nearly  doubling  the  maltose 
produced  per  unit  of  time.  The  maltose  in  turn  becomes  immedi- 
ately available  as  food  for  the  yeast  cells,  which  thereby  are  enabled  to 
renew  their  fermentation  activity  and  increase  the  rate  at  which  the 
CO2  is  produced  for  the  aeration  of  the  dough.  This  increasing  activ- 
ity is  further  augmented  by  a  temperature  increased  during  proofing 
of  the  panned  loaves;  this  furnishes  part  of  the  explanation  for  the 
rapid  rise  of  the  loaf  in  the  pan  just  before  baking.  The  yeast  activity 
is  likewise  stimulated  by  the  same  increase  in  temperature  and  acid- 
ity. That  it  is  the  function  of  the  diastatic  enzymes  present,  and  not 
of  the  sugar  originally  added  to  the  dough,  which  largely  controls  the 
proofing  action,  is  shown  further  on  in  connection  with  experiments 
on  the  diastatic  action  in  actual  doughs. 

We  should  not  lose  sight  of  another  important  factor  in  the  latter 
stages  of  fermentation  and  proofing  of  the  dough.  That  is  the  soft- 
ening effect  of  the  proteoclastic  agents  on  the  gluten.  The  gluten 
suffers  more  or  less  rapid  proteolysis :  becomes  softer  and  less  elastic. 
Thus  the  increasing  rate  of  carbon  dioxide  production  is  able  to 
"raise"  the  dough  easier  and  more  quickly. 

Buffer  Action.  The  buffer  effect  of  different  flours  must  likewise 
be  taken  into  consideration  in  this  connection.  The  highly  refined 
flours,  such  as  patents,  generally  show  a  lower  buffer  value  than  the 
less  highly  refined,  or  clear  flours.     Therefore,  the  maximum  acidity 

56 


should  be  reached  more  quickly  during  the  fermentation  of  a  patent 
flour,  resulting  in  a  more  rapidly  increasing  rate  of  fermentation  and 
consequently  a  better  aeration.  On  the  other  hand  the  better  quality 
of  gluten  in  these  grades  of  flour  requires  more  time  for  its  proper 
"ripening,"  is  more  tenacious  and  elastic,  and  so  requires  a  greater  fer- 
mentation activity  to  overcome  its  tightness.  Also,  its  greater  gas 
retaining  capacity  helps  to  make  up  a  combination  of  strength  char- 
acteristics which  taken  altogether,  form  the  desirable  qualities  for  the 
production  of  a  good  loaf  of  bread. 

The  flours  of  poorer  grade,  such  as  high  percentage  straights  and 
the  clears,  though  made  from  the  same  wheat,  usually  show  a  higher 
bufTer  value.  The  natural  effect  of  their  buffer  salts  is  therefore  to  re- 
quire a  greater  amount  of  acid  formed  by  fermentation  before  the 
proper  pH  of  the  resulting  dough  is  reached  for  maximum  diastatic 
activity.  It  may  be  that  the  same  concentration  of  hydrogen-ions, 
namely  10'^  is  not  reached  in  the  doughs  from  low  grade  flours.  The 
decreasing  quality  of  the  glutens,  however,  in  the  lower  grades  of 
flours,  is  oftener  the  limiting  factor  because  of  their  very  poor  gas  re- 
taining capacity,  arid  they  are  able  to  show  but  little  improvement  by 
the  addition  of  acids  and  diastatic  enzymes.  Thus  before  the  inter- 
relationships of  these  three  factors,  gluten  strength,  proteoclastic 
activity,  and  diastatic  activity  can  be  more  accurately  explained,  it 
becomes  necessary  for  further  study  on  each  one  of  them. 

The  above  consideration  of  buffer  values  of  flour  and  hydrogen-ion 
concentrations  in  the  dough  demonstrate  the  desirability  of  deter- 
mining these  values  for  each  of  the  flour  samples  used  for  this  investi- 
gation. The  results  of  such  a  set  of  determinations  are  given  in  part 
here,  and  afford  a  confirmation  of  those  of  Bailey  and  Peterson  (1921). 
The  buffer  values  of  the  fourteen  flours  were  determined  by  making 
water  suspensions  of  each  sample,  of  1  :  5  concentrations,  as  recom- 
mended by  Bailey  and  Peterson.  These  were  kept  at  25. °C  and 
shaken  at  intervals  for  one  hour.  The  suspensions  were  then  centri- 
fuged  without  filtering,  and  to  25.  cc  aliquots  of  the  centrifugate  were 
added  varying  amounts  of  .02  Nor/nal  HCl  or  NaOH.  The  resulting 
pH  was  determined  at  once  in  Bailey  electrodes  by  potentiometer 
measurements.  For  the  sake  of  brevity  only  one  of  the  fourteen  sets 
of  readings  is  included  in  Table  XVII.  Figure  4  includes  the  graphs 
of  three  sets  of  values  of  widely  different  grade.  The  addition  of  .02N 
acid  was  carried  up  to  40.  cc  to  better  show  the  differences  in  the 
shapes  of  the  curves. 


57 


TABLE  XVII. 


The  Buffer  Value  of  Flour  Sample  No.  1001. 


cid  or  Alkal 

I  Added  to  25  cc's 

of  1.5 

Flour  Extract 

12.5 

cc. 

.02N 

HCl 

10.0 

cc 

.02N 

HCl 

7.5 

cc 

.02N  HCl 

5.0 

cc 

.02N 

HCl 

2.5 

cc 

.02N 

HCl 

0.0 

cc 

2.5 

cc 

.02N 

NaOH 

5.0 

cc 

.02N 

NaOH 

7.5 

cc 

.02N 

NaOH 

10.0 

cc 

.02N 

NaOH 

12.5 

cc 

.02N 

NaOH 

pH. 

2.519 
2.654 
2.925 
3.388 
4.150 
5.816 
7.371 
9.045 
9.775 
10.253 
10.617 


cc   IficWI 


Ct%o* 


Figure  4. 

The  buffer  curves  of  three  flour  of  widely  different  baking  value, 

acid  range. 

58 


Because  of  the  large  number  of  buffer  values  falling  within  rather 
narrow  limits,  and  the  crossing  of  the  curves  on  the  alkaline  side,  the 
complete  curves  cannot  all  be  drawn  in. 

Figure  V  affords  a  comparative  idea  of  the  buffer  values  in  the  acid 
range,  for  each  of  the  flours  used.  The  height  of  the  columns  repre- 
sent the  relative  change  in  pH  resulting  from  the  addition  of  20.  cc  of 
.02N  HCl  to  100  cc  of  the  centrifuged  1  :  5  flour-water  extract.  The 
arrangement  of  flours  is  in  the  order  of  their  baking  value.  For  con- 
venience the  series  of  fourteen  samples  have  been  arbitrarily  divided 
into  three  classes.  The  columns  in  black  represent  the  group  of 
flours  which  would  ordinarily  be  considered  by  the  baker  as  those 
possessing  good  baking  qualities.  The  crossed  lines  indicate  those 
flours  of  rather  poor  quality,  but  which  can  nevertheless  be  used  for 
bread,  and  especially  so  when  blended  with  stronger  flours.  The  col- 
umns in  diagonal  lines  represent  those  flours  which  are  of  such  poor 
quality  and  low  baking  value  that  they  could  not  be  used  for  market- 
able bread  in  this  country.  While  flour  No.  1013  might  be  included 
in  the  second  group  because  of  its  relative  strength,  its  poor  color 
would  make  it  of  doubtful  value  for  blending. 


^   ^    ^ 


Hard  Wheat  Patents 
and  Straight  Flours 


Soft        Clear  Grade 
Wheat  Flours 

Flours 


Figure  5 

The  relative  change  in  pH  produced  by  addition  of  20  cc  HCl  to 
100  cc  1 :5  flour-water  suspensions.  Flours  arranged  in  the  order  of 
their  baking  value. 

59 


There  is  no  apparent  relationship  shown  by  the  values  for  buffer 
action  and  the  corresponding  diastatic  activity  of  the  flour  as  given  in 
Table  XVIII.  This  could  only  be  shown  by  a  set  of  determinations  of 
both  pH  and  diastatic  activity  in  the  actual  dough  during  fermenta- 
tion. The  changes  of  pH  with  progressive  fermentation  of  the  doughs 
made  from  this  set  of  flours  are  given  in  another  report.  Work  is 
now  in  progress  in  several  quarters  on  the  development  of  a  special 
form  of  electrode  for  the  determination  of  hydrogen-ion  concentration 
in  doughs.  Experiments  in  the  laboratories  of  one  of  the  larger 
American  bakeries  have  demonstrated  the  commercial  application  of 
such  electrometric  measurements  for  the  control  of  dough  fermenta- 
tions. 

Relative  Diastatic  Powers  of  Flour  Samples.  The  results  on  deter- 
minations of  the  diastatic  powers  of  each  of  the  fourteen  flours  are 
given  in  Table  XVIII. 

The  regular  procedure  described  herein  was  used,  namely,  one  hour's 
autolysis  of  a  1  :10  flour-water  suspension  @  27°  centrigrade. 


TABLE  XVIII. 

Comparative  measurements  of  diastatic  power  on  fourteen  samples 

of  flour. 

Diastatic  Power 
Maltose   by- 
Diastase  in 
10.  g.  Flour 
Milligrams 
248.2 
186.5 
34.8 
145.0 
131.9 
105.7 
123.6 
304.1 
211.8 
92.6 
51.7 
132.9 
132.6 
116.2 


Active  Sample 

Inactive  Sample 

Flour 

Weighed  Cu^O 

Weighed  CuaO  per 

Sample 

per  2.5  g.   Flour 

2.5  g.  Flour 

No. 

Grams 

Grams 

1001 

.1031 

.0223 

1002 

.0762 

.0148 

1003 

.0226 

.0091 

1004 

.0638 

.0155 

1005 

.0626 

.0185 

1006 

.0513 

.0154 

1007 

.0563 

.0145 

1008 

.1146 

.0162 

1009 

.0833 

.0140 

1010 

.0474 

.0157 

1011 

.0254 

.0065 

1012 

.0529 

.0085 

1013 

.0523 

.0080 

1014 

.0553 

.0161 

The  relation  of  the  respective  diastatic  powers  of  these  flours  to 
their  baking  strength  can  be  more  easily  shown  in  Figure  6,  where  the 
arrangement  of  flours  is  according  to  baking  strength,  the  height  of 
column  representing  the  milligrams  of  maltose  produced  in  one  hour 
by  10.  grams  of  flour.  Each  value  is  the  average  of  three  or  more 
determinations  for  each  flour.  With  the  exception  of  the  very  low 
grade  flours  these  samples  show  diastatic  powers  which  are  a  fairly 


60 


good  index  of  their  general  fermentation  characteristics.  That  is,  the 
behavior  of  the  dough  in  the  latter  stages  of  fermentation,  and  the 
"spring"  in  the  oven,  show^  that  the  diastatic  power  of  the  flour  as 
measured  by  the  method  here  employed  does  furnish  an  indication  of 
the  general  strength  characteristics  of  that  flour,  especially  w^ith  regard 
to  volume  and  texture.  The  quality  of  the  gluten,  however,  must  be 
sufficiently  high  to  conserve  the  value  of  the  diastatic  action  and  make 
a  good  loaf  possible.  It  would  be  necessary  to  study  many  more 
samples  of  flour  before  extending  such  an  observation  to  the  status 
of  a  general  conclusion. 

From  these  samples  of  flour,  differing  so  widely  in  type  and  grade, 
no  conclusions  can  be  drawn  as  to  the  relationship  between  diastatic 
activity  of  a  flour  and  climatic  factors  in  the  growth  of  the  wheat. 
Only  flours  produced  by  a  uniform  milling  practice  from  wheats  typi- 
cal of  diff"erent  growing  regions  could  be  used  to  furnish  information 
of  such  a  nature. 


^00  — 


Hard  Wheat  Patents 
and  Straight  Flours 


£!>  c^      t\ 

Soft  Clear  Grade 
Wheat        Flours 
Flours 


Figure  6 
Relative  diastatic  powers  of  fourteen  flours,  in  order  of  their 

baking  value. 

61 


Diastatic  Activity  During  Fermentation  of  the  Dough.  The  opin- 
ions of  several  of  the  earher  investigators  concerning  the  significance 
of  diastatic  enzymes  in  a  dough  during  the  latter  stages  of  fermenta- 
tion have  been  revievv^ed  in  the  historical  part  of  this  paper. 

The  early  English  practice,  revived  during  the  late  war,  of  ferment- 
ing doughs  without  added  sugars  would  throw  the  burden  of  sugar 
production  entirely  upon  the  diastase  of  the  flour  unless  other  dias- 
tatic material,  such  as  malt,  were  added.  The  production  of  carbon 
dioxide  by  the  zymase  of  the  yeast  cells  must  continue  with  some 
regularity  throughout  the  frementation  period  in  order  that  the  dough 
mass  may  be  properly  aerated. 

The  increased  enzymatic  activity  during  the  forty-five  to  sixty 
minutes  in  which  the  dough  is  allowed  to  rise  in  the  pan  before  bak- 
ing is  of  especial  importance.  It  is  this  final  period  of  aeration  which 
determines  to  a  large  extent  the  texture  and  "lightness"  of  the  loaf. 
The  gluten,  however,  must  have  the  requisite  quality.  The  proofing 
period  is  usually  carried  out  at  a  temperature  of  eight  to  ten  degrees 
higher  than  that  of  the  fermentation  up  to  this  point.  It  is  observed 
from  Figures  1  and  2  that  this  increase  in  temperature  from  27°  to 
35°C  increases  the  diastatic  activity  nearly  30  percent.  In  actual 
baking  practice,  however,  the  rise  of  temperature  in  the  interior  of 
the  proofing  loaf  is  very  slow  and  a  temperature  of  33  to  35°C  is  prob- 
ably not  reached  until  near  the  end  of  the  proofing  period. 

If  the  yeast  cell  is  to  increase  its  zymase  activity  during  proofing 
in  order  to  provide  the  carbon  dioxide  necessary  to  properly  raise  the 
dough  and  shape  the  loaf,  there  obviously  must  be  a  sufificient  supply 
of  available  sugar.  If  the  present  commercial  practice  of  straight 
dough  fermentation  there  is  rarely  a  sufficient  supply  of  sugar  added 
at  the  time  of  rnixing  to  carry  the  yeast  activity  throughout  the  fer- 
mentation. The  diastase  of  the  flour,  if  present  in  sufficient  quan- 
tity, must  carry  a  part  of  the  load.  The  addition  of  a  fresh  supply 
of  sugar  by  mixing  in  with  the  dough  when  ready  for  proofing  is  not 
practical  for  several  reasons,  and  a  too  large  supply  at  the  time  of 
mixing  the  dough  often  stimulates  the  yeast  activity  far  beyond  that 
desired.  The  yeast  must  then  depend  largely  upon  the  sugars  produced 
by  the  diastase  for  its  fermentation  activity  during  the  proofing.  This 
sugar,  in  the  form  of  maltose,  is  readily  split  dow^n  to  dextrose  by  the 
maltose  of  the  yeast,  and  so  is  immediately  available  as  substrate  for 
the  yeast  zymase. 

We  might  also  expect  that  there  would  be  a  slight  difference  shown 
in  the  rate  of  carbon  dioxide  production  between  the  action  of  yeast 
fed  on  sucrose  added  at  mixing,  and  on  maltose  produced  by  diastase 
in  the  dough.     The  latter  may  be  regarded  as  a  steady  rate  of  maltose 

62 


formation  within  the  colloidal  medium  immediately  surrounding  the 
yeast  cells.  The  distance  the  maltose  must  diffuse  to  reach  the  ac- 
tive yeast  surface  is  thus  at  a  minimum.  On  the  other  hand  the  util- 
ization of  sugars  added  to  the  dough  must  decrease  in  rate  as  the  dif- 
fusion distance  from  the  active  yeast  interface  increases.  This  v^ould 
further  lead  to  the  opinion  that  in  theory  the  substitution  of  the  req- 
uisite diastatic  enzymes  for  a  part  of  the  sugar  usually  added  to  the 
dough  at  mixing  should  produce  a  better  and  more  uinform  fermenta- 
tion, and  in  general  a  better  loaf.  Some  sugar  of  course  should  be 
present  at  the  beginning  of  the  fermentation  to  stimulate  the  yeast 
activity  and  supply  food  for  the  early  fermentation,  unless  the  whole 
fermentation  period  be  lengthened  accordingly.  After  that  time  the 
diastase  should  have  reached  nearly  a  constant  rate  of  maltose  produc- 
tion and  should,  therefore',  be  able  to  supply  the  necessary  sugar  in  a 
more  available  form. 

In  practice,  however,  the  addition  of  considerable  amounts  of  dias- 
tatic malt  preparations,  e.  g.,  malt  flours  or  malt  extracts,  is  compli- 
cated by  the  excessive  activity  of  the  proteoclastic  enzymes  which 
they  usually  contain.  These  malt  preparations  likewise  often  contain 
pigmented  material  which  is  not  destroyed  during  fermentation.  In 
fact  some  of  the  pigmented  particles  appear  to  be  so  affected  by  fer- 
mentation that  they  are  made  soluble  and  tend  to  diffuse  into  the 
dough,  darkening  the  color  of  the  finished  product.  Here  again  the 
importance  of  the  proteoclastic  enzymes  and  the  necessity  for  their 
measurement  and  control,  often  overshadow  the  practical  application 
of  diastatic  malt  products  in  the  bakery. 

It  appeared  highly  desirable,  therefore,  to  make  some  measurements 
of  sugar  content  and  diastatic  activity  in  doughs  during  the  normal 
fermentation.  Regular  test  doughs  were  accordingly  mixed,  omitting 
either  the  yeast,  or  sugar,  or  both,  depending  upon  which  type  of  re- 
sultant sugar  content  was  to  be  measured.  The  lengthy  method  of 
extracting  the  sugars  from  these  doughs  with  alcohol,  followed  by 
evaporation,  dilution,  clarification,  etc.,  is  hardly  suited  to  the  rapid 
determination  of  total  and  reducing.«ugars  in  several  doughs  at  short 
intervals.  The  procedure  which  had  been  previously  used  for  the 
measurement  of  diastatic  activity  in  doughs  suggested  itself  as  a  possi- 
ble basis  for  a  satisfactory  method.  Several  trials  at  30  minute  inter- 
vals during  the  fermentation  of  a  dough  without  yeast,  gave  results 
which  w^hen  plotted  in  terms  of  sugars  produced  per  unit  of  time, 
showed  a  smooth  curve  corresponding  roughly  to  the  enzyme  curves 
of  Figure  2.  The  curves  for  a  number  of  flours  indicate  that  the 
method  will  at  least  give  comparative  results  for  each  type  of  sugar 
content  and  for  each  flour. 

63 


The  procedure  finally  used  for  these  determinations  was  as  follows : 
A  two-loaf  dough  was  mixed  as  for  a  baking  test  in  the  manner  pre- 
viously described.  This  was  taken  from  the  mixer  and  divided  into 
two  equal  batches,  one  of  which  was  fermented  and  baked,  and  used 
as  a  control  dough,  the  other  being  handled  in  the  same -manner  ex- 
cept that  from  it  samples  were  taken  at  desired  intervals.  From 
these  samples  two  ten  gram  portions  were  weighed  off,  one  taken  im- 
mediately for  sugar  analysis,  and  the  other  used  for  the  determination 
of  total  solids.  The  sample  to  be  analyzed  for  sugars  was  at  once 
shaken  up  in  a  500  cc  bottle,  with  about  100  cc  of  distilled  water  to 
which  two  or  three  drops  of  50%  NaOH  had  been  added,  until  the 
gluten  had  been  dispersed  and  the  sugars  dissolved  out.  This  dough 
suspension  was  poured  into  a  300  cc  volumetric  flask,  clarified  and 
treated  as  described  above  in  the  regular  procedure.  In  some  cases  a 
50.  cc  aliquot  of  the  clear  centrifuge  was  taken  for  hydrolysis  and 
determination  of  total  sugars  by  the  A.  O.  A.  C.  method.  The  results 
for  the  total  sugars  while  substantiating  the  conclusions  to  be  drawn 
from  those  of  the  reducing  sugars,  do  not  add  materially  to  the  infor- 
mation desired,  and  are  not  included  here,  as  they  were  not  obtained 
in  complete  sets  for  all  samples. 

Many  determinations  were  made  in  this  laboratory  on  the  effects  of 
diastatic  action  during  the  fermentation  of  doughs.  Different  kinds 
of  diastatic  malt  preparations,  in  varying  amounts  were  used  in  the 
doughs,  with  and  Without  other  sugars,  and  with  and  without  yeast. 
The  results  are  included  in  a  separate  report  on  flour  strength  as  in- 
fluenced by  the  addition  of  diastatic  ferments.  (Collatz,  1922).  Only  a 
few  examples  have  been  selected  from  this  available  data  to  show  the 
role  which  diastase  plays  in  panary  fermentation.  These  are  designated 
by  Roman  numerals  from  I  to  VIII,  corresponding  to  the  curves  in 
Figures  7-10.  The  experiments  with  these  doughs  were  made  in  pairs, 
one  dough  of  each  pair  was  made  up  with  the  usual  amount  of  yeast, 
the  other  without  yeast.  In  this  manner  the  relation  of  sugar  pro- 
duced by  the  diastase  to  that  required  by  the  yeast  fermentation 
under  the  same  conditions  to  temperature  is  clearly  shown.  Dough  I 
was  the  regular  baking  test  dough,  containing  3.0%  of  sucrose  and 
2.5%  yeast.  The  resulting  loaf  was  above  the  average,  though  slightly 
overproofed.  The  texture  was  fair,  the  volume  large  (2110.  cc),  and 
the  color  good,  but  the  grain  was  a  bit  too  open  because  of  overproof. 
Dough  II  was  the  same  as  No.  I  except  that  the  yeast  had  been 
omitted.  Dough  III  was  mixed  without  sugar,  but  otherwise  the 
same  formula  as  Dough  I.  The  fermentation  time  was  necessarily 
.longer.  The  resulting  loaf  was  of  very  good  quality,  of  very  fine 
grain,  good  texture  and  an  even  but  strong  break.     The  color  of  crust 

64 


was  fair,  but  considerably  lighter  than  that  of  loaf  I,  as  would  be 
expected  from  the  lack  of  sugar.  The  volume  likewise  was  less,  be- 
ing only  1870.  cc.  Dough  IV  was  the  same  as  No.  Ill,  omitting  the 
yeast.  The  supply  of  flour  No.  1009  was  exhausted  at  this  point  and 
another  sample  was  obtained  from  the  same  mill.  This  second 
sample,  designated  as  No.  1009A  was  somewhat  inferior  in  strength 
and  color,  but  the  behavior  of  its  diastatic  enzymes  were  practically 
the  same  as  those  of  No.  1009.  Diastatic  activity  and  reducing  sugar 
determinations  for  doughs  mixed  with  and  without  sucrose  showed 
practically  the  same  curves  as  those  for  doughs  Nos.  1  to  IV.  Flour 
No.  l609A  was,  therefore,  used  for  doughs  V  to  VIII.  Dough  V 
was  mixed  with  2.%  of  a  malt  flour  as  a  source  of  added  diastatic  en- 
zymes, but  no  other  sugar  was  added.  Although  the  amount  of  this 
particular  malt  flour  was  somewhat  in  excess,  as  indicated  by  the 
grayish  color  of  the  crumb  and  the  slight  coarsening  of  the  grain,  the 
effect  of  the  added  enzyme  is  clearly  shown  in  curve  V.  The  volume 
of  the  loaf  was  increased  12%  over  that  of  a  standard  dough  contain- 
ing sucrose  but  no  added  malt  diastase.  The  further  discussion  of 
this  phase  of  the  question  of  added  diastatic  enzymes  is  considered  in 
detail  in  the  second  report.  (Collatz,  1922).  Dough  VI  was  mixed  with 
the  2.%  of  malt  flour  but  contained  no  yeast.  Dough  VII :  The  total 
amount  of  maltose  produced  in  dough  V  by  diastatic  action  of  com- 
bined wheat  flour  and  malt  flour  was  calculated  back  into  terms  of 
anhydrous  maltose  per  unit  weight  of  flour.  This  percentage  of  an- 
hydrous c.p.  maltose  was  then  added  to  Dough  VII,  without  other 
sugars  or  diastatic  products.  The  resulting  loaf  bore  the  same  rela- 
tion to  the  standard  loaf  as  in  the  case  of  Doughs  Nos.  I  and  II, 
namely,  a  finer  grain,  good  texture,  lighter  color  of  crust,  and  slightly 
smaller  volume.  Table  XIX  is  furnished  here  as  an  example  of  the 
data  obtained  for  each  dough,  and  comprises  the  basis  from  which 
the  points  were  plotted  in  Curve  III,  Figure  8.  To  save  space  and 
to  avoid  multiplicity  of  headings  the  data  for  the  complete  set  of 
curves.  Figures  8,  9,  10  and  11,  are  abbreviated  and  grouped  in  Table 
XX. 


65 


TABLE  XIX. 

Reduc'ng  Sugars  and  Diastatic  Activity  During  the  Fermentation  of 
a  Dough.    Mixed  Without  Sugar.  Curve  III. 


Weight   of 

Reducing 

Total 

Weight   of 

Sugars  as 

Solids 

CU2O  per 

Maltose 

Sampled 

Total 

Temper- 

Weight of 

per  10.  g. 

10.    g. 

per  10.  g. 

At 

Time 

ature 

Dough 

Dough 

Dough 

Dough 

Minutes 

Centigrade 

Grams 

Grams 

Milligrams 

Milligrams 

Mixing 

0.0 

27.° 

530.0 

5.5720 

207.0 

156.0 

1.    hour 

60.0 

27.5° 

274.4 

209.5 

1st  Punch 

177.0 

27.5° 

526.0 

5.5222 

269.4 

205.5 

2d  Punch 

252.0 

27T 

522.0 

5.5086 

197.4 

148.3 

To  Bench 

297.0 

28.5° 

519.0 

5.5261 

168.0 

125.2 

To  Proof 

312.0 

To  Oven 

359.0 

36.0° 

5.5476 

116.8 

84.6 

Out  Oven 

377.0 

(226.5°) 





187.6 

140.5 

TABLE  XX. 

Weights  of  Reducing  Sugars  Calculated  as  Milligrams  of  Maltose  in 
Ten  Grams  of  Dough  at  Different  Stages  of  Fermentation. 

Straight  Dough     Straight  Dough  Xo  Sugar  Straight  Dough 

2.5%  Sucrose  No  Sugar  2%  Malt  Flour       1.837^'  Maltose 


I. 

Sampled  With 
At:        Yeast 

II. 

Without 

Yeast 

III. 
With 
Yeast 

IV. 

Without 

Yeast 

V. 

With 

Yeast 

VI. 

W thout 
Yeast 

VII. 

With 
Yeast 

VIII. 

Without 

Yeast 

Mixing 

481.9 

99.9 

156.0 

125.7 

231.6 

184.6 

231.0 

200.9 

1   Hour 

First 

Punch 

Second 

Punch 

To 

Bench 

To 

Proof 

To 

Oven 

Out  of 

Oven 

456.6 
395.8 
274.1 

ISl.O 
178.2 
240.2 

209.5 
205.5 
n8.3 

159.4 
204.1 
222.0 

307.9 
278.2 
308.6 

235.7 
286.5 
326.1 

288.7 
279.3 
261.3 

230.8 
288.9 
306.0 

222.6 

260.2 

125.2 

233.6 

254.2 

312.4 

186.8 
209.4 

256.6 
624.1 

"84.6 
140.5 

245.3 

333.8 
347.8 
258.7 

364.3 
36S.0 

211.9 
272.8 

320.6 

The  conclusions  to  be  drawn  from  this  set  of  results  in  Table  XX 
are  verified  by  considerable  additional  data  obtained  on  other  flours, 
as  included  in  the  second  report.  Curve  I,  Figure  7,  shows  the  rapid 
consumption  of  reducing  sugar  by  the  yeast  zymase  in  the  production 
of  carbon  dioxide  and  alcohol.  The  fall  of  reducing  sugar  content 
slows  up  about  the  time  the  dough  goes  to  the  proof-cabinet,  indicat- 
ing that  the  increased  diastasis  due  to  increased  temperature  is  mak- 
ing itself  felt.  This  latter  increase  is  shown  nicely  by  Curve  II.  The 
wide  difference  between  the  reducing  sugars  present  in  the  two  doughs 

66 


is  of  significance,  considering  that  in  Dough  I  the  sugar  was  added 
as  non-reducing  sugar  (sucrose).  This  must,  therefore,  be  ascribed 
in  part,  if  not  entirely,  to  the  rapid  hydrolysis  of  sucrose  by  the  inver- 
tase  of  the  yeast  cells.  Otherwise  it  would  likewise  appear  in  Curve 
II.  In  other  words,  the  yeast  cell  appears  to  invert  the  sucrose  faster 
than  its  zymase  activity  requires.  This  same  result  is  observed  in 
every  case  where  this  sort  of  determination  has  been  made.  In  the 
other  pairs  of  curves  the  divergence  is  not  so  great  because  the 
amount  of  disaccharides  present  are  much  smaller. 

Curve  III,  Figure  8,  emphasizes  the  fact,  as  indicated  by  Curve  I, 
that  the  yeast's  requirement  of  available  sugars  continues  throughout 
fermentation,  and  really  increases  with  the  stimulated  activity  during 
the  proofing  of  the  loaf.  In  this  curve  the  yeast  zymase  has  practic- 
ally exhausted  the  supply,  and  the  volume  of  the  baked  loaf  suffers  in 
consequence,  even  though  the  flour  diastase  (Curve  IV)  is  still  active. 
Curves  V  and  VI,  Figure  9,  on  the  other  hand,  illustrate  an  entirely 
different  state  of  affairs.  With  a  sufficient  supply  of  diastatic  power 
the  excess  of  reducing  sugars  (above  the  yeast  requirement)  instead 
of  falling  off  after  three  hours  of  fermentation  as  in  the  two  previous 
cases,  is  carried  along  in  an  upward  curve  until  it  is  no  longer  needed. 
The  result  is  an  increase  in  loaf  volume.  When  this  added  diastase 
can  be  supplied  w^ithout  the  associated  undesirables  of  coloring  matter 
and  excessive  proteoclastic  action,  there  should  be  no  question  as  to 
its  importance  in  the  manufacture  of  good  bread.  Curves  VII  and 
VIII,  Figure  10,  show  much  the  same  situation  as  those  for  I  and  II. 
The  maltose,  as  sugar  added  at  mixing,  does  not  appear  to  sustain  the 
fermentation  in  its  later  stages  any  better  than  sucrose.  The  fermen- 
tation curve,  so-called,  does  appear  to  be  more  uniform,  but  whether 
that  difference  depends  upon  the  nature  of  the  sugars  is  still  to  be 
determined. 

One  more  point  remains  to  be  noted.  The  sudden  increase  of  re- 
ducing sugars  during  the  first  few  minutes  in  the  oven  is  shown  by 
the  dotted  lines  at  the  end  of  curves  I,  II  and  VII.  The  sudden  in- 
crease of  heat  in  the  oven,  decreasing  in  rate  from  the  exterior  to  the 
interior  of  the  loaf,  during  w^hat  the  baker  terms  the  "oven  spring," 
should  be  expected  to  steadily  raise  the  diastase  up  to  and  beyond  its 
optimum  temperature.  The  resulting  sugars  produced  in  the  loaf 
must  then  be  taken  into  consideration  in  the  interpretation  of  carbohy- 
drate anaylsis  of  bread. 


67 


1    r~i    1    \    \    I    I    1    I    1 — I — I — 1 — I — 1 — \ — I — \ — r 


J I I L 


X  60  ^90 

lime  -  minutes 


J \ I L 


ISO 


J I \ L 


no 


2fO 


J \ I [ I L 


Figure  7. 
Reducing  Sugars  Found  in  Doughs  During  Fermentation.     Curve  I. 

Straight  Dough.    Standard  Formula.    Curve  II. 
Straight  Dough.    Yeast  Omitted. 

1     \    \    I    I     I    I    \    I    \    1    ]    I    \    \    I    I    I    \    \ — \ — I — r 


iOC-^ 


30t- 


2M- 


I        J         I         f 


J \ \ 1 L_L 


i [ \ L 


^ 


J L 


30  60 

Time 


so  izo 

minutes. 


3V> 


360 


Figure  8. 
Reducing  Sugars   Found  in  Doughs   During   Fermentation.      Curve 
III.     Straight  Dough.     Sugar  Omitted.     Curve  IV.  > 

Straight  Dough.    Yeast  and  Sugar  Omitted. 

68  _ 


1 — \ — I — \    \    I    I    \    r 


1    1    I    \    \    r~i    r~i    I    ]    r 


r 


J \ \ \ I      I      I      I      I 


30  60  90 

Time  -  nunutes. 


J 1 \ L 


J 1 L 


Figure  9. 

Reducing  Sugars  Found  in  Doughs  During  Fermentation.    Curve  V. 

Straight  Dough.    Diastatx  Malt  Added.    Curve  VI. 

Straight  Dough  With  Added  Diastase, 

But  Yeast  Omitted. 

"1    \    \    \    \    i~~i~i    \    \    r~i    [    I    1    v~\    \    1    r-^ — 1 — r 


500- 


\ I \ L_J I \ 1 \ I     .1      I      i.     i 


30-60  ^90 

Time  -  minutes 


2/0 


149 


Figure  10. 

Reducing  Sugars  Found  in  Doughs  During  Fermentation.    Curve  VII. 

Straight  Dough.    Maltose  Substituted  for  Sucrose. 

Curve  VIII.    Straight  Dough.    Maltose 

Substituted,  Yeast  Omitted. 


69 


The  Relation  of  Diastatic  Power  to  Different  Forms  of  Starch.  I'e- 
fore  the  various  diastatic  preparations,  such  as  barley  malt  flours  and 
malt  extracts,  can  be  used  intelligently  in  panary  fermentation  to  sup- 
ply the  required  diastatic  power,  it  is  necessary  to  have  some  ra- 
tional basis  for  measurement.  The  diastatic  power  of  these  products 
Fhould  be  expressed  in  terms  of  their  ability  to  produce  maltose  per 
unit  weight  used,  under  the  conditions  of  baking  practice.  It  be- 
comes necessary  first  to  find  an  easily  standardized  substrate  which 
will  be  comparable  to  the  flour  which  goes  into  the  dough.  The  more 
accurate  way  of  determining  the  diastatic  activity  of  a  malt  product 
upon  the  particular  flour  with  w^hich  it  is  to  be  used  offers  many  ob- 
jections, both  technically  and  as  a  basis  for  control  and  commerce. 
The  use  of  wheat  flour  as  substrate  involves  the  determination  of 
the  diastatic  power  of  the  flour  itself,  the  condition  or  resistance  of  the 
starch  granules  to  the  enzyme,  the  acidity  and  buffer  value  of  the 
flour;  in  short,  as  many  factors  as  there  are  flours. 

The  next  possibility  is  in  the  selection  of  a  standard  starch  as  sub- 
strate, under  controlled  conditions  of  acidity  and  salt  concentration, 
which  will  give  results  comparable  to  average  bake  shop  conditions. 
Preliminary  experiments  have  furnished  some  data  which  are  given 
here  because  of  their  general  bearing  on  this  phase  of  the  problem. 

Five  forms  of  starch  were  used  in  this  work.  Starch  sample  num- 
ber 1,  was  the  starch  as  it  naturally  occurs  in  flour  sample  No.  1009, 
with  its  associated  diastase,  and  other  enzymes,  salts,  and  organic  ma- 
terials. Number  2  was  a  sample  of  Merck's  "soluble  starch  according 
to  Lintner,"  used  in  water  suspension  without  heating.  No.  3  was 
the  same  soluble  starch  gelatinized  by  boiling.  Sample  No.  4  was 
commercially  prepared  wheat  starch.  Sample  No.  5  was  a  sample 
of  wheat  starch  prepared  in  this  laboratory  as  previously  described. 

The  researches  of  Thatcher  and  Koch  (1914),  Thatcher  and  Ken- 
nedy (1920),  and  Sherman,  et  al.  (1913-1921),  furnish  examples  of 
the  use  of  soluble  starch  a-s  substrate  for  diastatic  enzymes.  The  con- 
tribution of  Swanson  and  Calvin  (1913),  showed  the  eft'ect  of  autolytic 
diastasis  on  natural  wheat  starch.  The  differences  betw^een  these  two 
forms  of  substrate  for  diastatic  activity  are  obvious  from  the  pub- 
lished data.  To  determine  whether  the  diastase  in  wheat  flour  would 
act  more  readily  on  added  soluble  starch  than  on  the  unchanged  starch 
granules  of  the  wheat  itself,  the  following  experiment  was  carried  out. 

One  10  gram  sample  of  flour  No.  1009  was  digested  with  100  cc  of 
water  for  one  hour  at  27° C  in  the  regular  manner.  To  a  second 
sample  were  added  10  grams  of  soluble  starch  (No.  2),  and  handled 
in  the  same  manner.  Blanks  w^ere  run  for  both  samples  to  determine 
their  natural  reducing  values.     The  results  are  given  in  Table  XXI, 

70 


and  are  the  averages  of  three  or  more  determinations  on  each  sample. 
There  is  a  considerable  augmentation  of  the  maltose  produced  by  the 
addition  of  a  more  easily  digested  form  of  starch  to  the  diastatic 
medium.  The  slight  increase  in  acidity  due  to  the  addition  of  the 
more  acid  soluble  starch  might  account  for  a  part  of  the  difference,  as 
shown  l)y  the  data  in  Table  XXII. 


TABLE  XXI. 

The  Activity  of  Wheat  Flour  Diastase  on  Wheat  Flour  Starch 

and  on  added  Soluble  Starch. 

Difference    Cal- 

Actual  Weight  of  culated  as  Mal- 

CuzO  per  50.  cc  tose  by  Dias- 

aliquot    of  tase  in  10. 

Solution  g.  Flour 

Starch   Used                                      Milligrams  Milligrams 

10.  g.  Starch  No.  1  (Wh't  flour  No.  1009  only)                82.3\  2O8  2 
10.  g.  Starch  No.  1   (Blank)                                               14.1  J 


10.  g.  Starch  No.  1  +  10.  g.  Starch  No.  2  114.9  \ 

10.  g.  Starch  No.  1-f  10.  g.  Starch  No.  2  (Blank)      25.6  J 


275.0 


The  effect  of  several  different  forms  of  starch  on  the  activity  of  a 
diastatic  malt  extract  w^as  next  tried.  There  v^^as  at  hand  for  this 
Mrork  a  fresh  sample  of  a  commercial  diastatic  malt  syrup  v^hich  had 
just  been  analyzed  for  total  solids,  ash,  and  proteins,  total  reducing 
sugars,  and  the  Lintner  value  of  which  v^^as  determined  as  44.4. 

The  starches  used  as  substrate  vi^ere  w^eighed  out  in  10  gram  samples 
into  300  cc  erlenmeyer  flasks  and  shaken  up  with  100  cc  of  water.  To 
these  there  were  added  10  cc  of  a  freshly  prepared  5%  solution  of  the 
malt  extract.  These  were  allowed  to  digest  one  hour  at  27°C,  and 
then  treated  in  the  regular  manner.  Because  of  the  high  acidities  of 
some  of  the  digestion  samples  they  were  repeated,  using  a  solution 
of  K2HPO4  and  KH2PO4  as  buffer  salts  to  control  the  pH.  For  con- 
venience the  two  sets  of  experiments  are  grouped  together  in  table 
XXII  to  show  the  amounts  of  maltose  produced  by  the  same  amounts 
of  diastase  under  different  conditioj:is. 

An  inspection  of  the  pH  values  in  Table  XXII,  considered  in  rela- 
tion to  the  graph  in  Figure  3,  shows  that  the  points  obtained  lie  for 
the  most  part  on  either  side  of  the  optimum  range,  and  a  slight  change 
toward  neutrality  should  result  in  a  considerable  increase  of  maltose 
production.  These  values  for  the  pH  are  likewise  rather  far  from 
those  which  normally  obtain  in  a  dough,  with  the  exception  of  Starch 
No.  2.  Consequently  the  maltose  produced  by  the  diastase  under 
these  conditions  cannot  be  taken  as  a  comparative  measurement  of 
diastatic  powier  of  the  malt  extract  in  a  dough. 

71 


TABLE  XXII. 

Maltose  produced  by  the  same  weight  of  Malt  Diastase  acting 

on  different  Starches  for  one  hour  at  27 °C. 


Starch  used  as  Substrate 
No.  1.  (Flour  1009)  Sample 
No.  1.  (Flour  1009)  Blank 
No.  1.  (Flour  1009  without  added  malt) 

(Sample) 

No.  1.  (Flour  1009  without  added  malt) 

(Blank) 


pH 
at  end  of 
Digestion 


5.801 


Weight  of  CuaO  Maltose  Pro- 


per 50  cc  of 
Digestion 
Solution 

Grams 

.25221 
.1284J 

.0823* 

.0141* 


duced  by- 
Diastase  of 
1  gram  Malt 

Extract 
Milligrams 

247.0 


No.  2.  (Soluble  Starch)    Sample  5.204  .1584\ 

No.  2.  (Soluble  Starch)    Blank  .1272J 

No.  2.  (Soluble  Starch  +  Buffers) 

Sample  6.847  .2183\ 

No.  2.  (Soluble  Starch  +  Buffers)  .1772/ 

Blank 


200.8 
561.4 


No.  3.  (Gelatinized  Starch)  Sample 
No.  3.  (Gelatinized  Starch)  Blank 


.4119x2\ 
.0652x2/ 


2759.4 


No.  4.  (Com'l  Wheat  Starch)  Sample  4.108 

No.  4.  (Com'l  Wheat  Starch)  Blank  1.014 

No.  4.  (Commercial  Wheat  Starch  + 

Buffers)  Sample  6.707 

No.  4.  (Commercial  Wheat  Starch  + 

Buffers)  Blanlc 


.1492\ 
.1218/ 

.1458"! 


157.8 
136.0 


No.  5  /Prepared  Wheat  St'ch)  Sample       4.924 
No.  5.  (Prepared  Wheat  Starch)  Blank 
No.  5.  (Prepared  Wheat  Starch  -f- 

Buffers)  Sample  7.098 

No.  5.  (Prepared  Wheat  Starch  + 

Buffers)  Blank 

♦(Values  taken  from  Table  XXI  to  furnish 
activity  of  the  flour  No.  1009.) 


.1470\ 
.1105/ 

.1324\ 
.1105/ 


215.2 


116.8 


a  correction   for  the  diastatic 


On  the  other  hand,  the  similarity  in  result  between  Starch  No.  1 
with  a  pH  of  5.8  or  less,  Starch  No.  2  with  a  pH  of  5.2,  and  of  Starch 
No.  5  with  a  pH  at  4.9,  would  indicate  the  probable  satisfactory  solu- 
tion of  the  problem.  The  results  for  Starch  sample  No.  3  confirm  the 
statements  as  reviewed  earlier  in  this  report.  There  can  then  be  no 
question  as  to  the  fallacy  of  reporting  the  diastatic  power  of  a  malt 
product  for  bread  making  in  terms  of  its  action  on  gelatinized  soluble 
starch.  It  is  hoped  that  a  completed  report  can  be  made  on  this  phase 
of  the  work  at  an  early  date. 


72 


Additional  analytical  data  on  each  of  the  fourteen  flour  samples 
used  in  these  experiments  is  compiled  for  reference,  and  is  appended 
as  Table  XXIII. 

TABLE  XXIII. 

Analytical  Data  ori  Flour  Samples. 

Acidity 
As  Lac- 


Sample 

Moisture 

Ash 

Protein 

Wet 

Dry 

tic 
Acid, 

PH 

Baking 
Value 

No. 

% 

% 

% 

% 

% 

% 

1009 

11.61 

.423 

13.81 

31.21 

11.18 

.20 

5.981 

100 

1001 

12.15 

.405 

11.34 

35.21 

11.09 

.14 

5.816 

99 

1008 

11.70 

.459 

15.32 

44.65 

15.44 

.20 

6.133 

97 

1002 

12.14 

.610 

13.00 

41.97 

13.28 

.16 

6.052 

95 

1012 

12.72 

.407 

12.76 

38.42 

12.94 

5.732 

91 

1006 

11.96 

.378 

11.96 

35.49 

12.96 

.135 

5.777 

91 

1005 

11.27 

.431 

11.04 

29.15 

11.15 

.14 

5.843 

90 

1010 

12.48 

.539 

10.36 

30.77 

10.99 

.18 

5.961 

83 

1011 

11.43 

.562 

10.77 

29.63 

10.71 

.22 

6.05 

76 

1003 

13.06 

.463 

8.83 

25.13 

9.22 

.11 

6.044 

63 

1013 

12.83 

.543 

14.83 

44.55 

15.63 

5.596 

56 

1007 

11.06 

.637 

14.12 

36.58 

14.18 

.20 

6.132 

46 

1004 

10.58 

.829 

12.70 

29.58 

12.11 

.26 

6.166 

35 

1014 

12.22 

.795 

14.37 

39.96 

14.47 

5.843 

32 

SUMMARY 


Diastatic  enzymes  are  recognized  as  one  of  the  important  factors 
which  go  to  make  up  "flour  strength." 

Experiments  to  "define  more  clearly  the  action  of  diastase  in  the 
production  of  better  bread  are  indicated  by  a  review  of  the  literature 
dealing  with  the  different  factors  of  flour  strength. 

The  effects  of  diastatic  enzymes  of  the  wheat  flour  in  panary  fer- 
mentation, with  respect  to  concentration,  time,  temperature,  acidity, 
and  diastatic  power,  have  been  determined. 

Representative  flour  samples,  numbering  fourteen  in  all,  were  col- 
lected from  typical  wheat  growing  regions  of  North  America  to  serve 
as  a  basis  for  the  study  of  diastatic  enzymes  in  bread  making. 

The  baking  values  of  the  flour  samples  were  determined  by  means 
of  comparative  baking  tests.  Other  available  analytical  data  has 
been  tabulated  along  with  these  baking  values  in  order  to  correlate 
as  many  different  factors  as  possible  with  the  "strength"  of  each  flour. 

11 


Protein  precipitation  from  cereal  extracts  by  the  tungstic  aciH 
reagent  of  Folin  and  Wu  was  studied  in  its  application  to  the  deter- 
mination of  diastatic  power  in  flour,  A  convenient,  rapid,  and  com- 
plete removal  of  proteins  from  solution,  without  filtration,  is  effected 
by  the  use  of  3.  cc  of  15%  sodium  tungstate  per  10  grams  of  flour, 
acidifying  with  sulfuric  acid  to  a  pH  of  2,  with  subsequent  centrifug- 
ing.     Diastatic  activity  is  completely  inhibited  by  this  treatment. 

Diastatic  powers  of  fourteen  flour  samples  were  determined  bv  on*^^" 
hour  autolytic  digestion  at  27°C.  The  maltose  produced  serves  as 
a  measure  of  the  diastatic  activity  of  that  flour  when  in  the  form  of 
a  dough. 

Maximum  activity  of  flour  diastase  in  a  dough  at  any  given  tem- 
perature or  pH  is  not  reached  at  once  because  of  insufiicient  w'ater. 
The  slower  hydration  of  the  colloidal  materials  compensates  in  some 
measure  for  the  more  rapid  loss  of  activity  which  the  diastase  would 
suffer  in  water  suspension,  and  hence  the  rate  of  maltose  production 
in  the  dough  is  quite  regular  at  any  given  temperature. 

Optimum  hydrogen  ion  concentration  for  flour  diastase,  pH  of  4.7, 
is  seldom  reached  during  the  fermentation  of  a  normal  dough.  The 
increase  of  acidity  during  fermentation,  in  the  range  of  pH  6  to  5,  has 
the  effect  of  considerably  increasing  the  production  of  maltose  toward 
the  latter  part  of  fermentation.  The  effect  of  hydrogen  ion  concen- 
tration on  the  activity  of  flour  diastase  is  in  agreement  with  that 
found  by  Sherman  and  others  for  the  diastase  of  malt. 

Temperature  is  the  most  important  factor  in  the  control  of  diastatic 
action  in  the  dough.  The  increased  temperature  at  proofing,  along 
with  the  increased  hydrogen  ion  concentration,  combine  to  make  the 
effect  of  diastase  most  significant  during  the  later  stages  of  fermenta- 
tion. It  is  during  this  later  period  that  the  diastase  produces  the 
necessary  sugars  from  which  the  yeast  may  complete  the  aeration 
of  the  dough  during  the  proofing  of  the  loaves. 

The  flour  showing  the  greater  diastatic  power  should  show  the 
greater  strength  and  consequently  the  greater  baking  value,  providing 
the  relative  quality  and  quantity  of  the  gluten  is  the  same. 

A  rational  standard  method  is  needed  for  measuring  the  diastatic 
materials  such  as  are  in  general  use  in  bread  making.  Experiments 
arc  described  which  demonstrate  the  necessity  for  using  a  standard 
starch  substrate,  and  a  possible  method  for  preparing  such  a  sub- 
strate is  suggested. 

74 


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84 


BIOGRAPHICAL. 

Louys  A.  Rumsey  was  born  May  7th,  1889,  at  Stryker,  Ohio.  He 
attended  the  public  schools  and  graduated  from  the  high  school  there 
in  1908.  Entering  Denison  University,  Granville,  Ohio,  in  the  fall  of 
1908,  he  specialized  in  chemistry  and  graduated  with  the  B.  S.  degree 
in  June,  1912.  He  returned  to  Denison  the  following  year  and  re- 
ceived the  M.  S.  degree  in  June,  1913.  The  thesis  work  was  done  in 
the  department  of  chemistry  on  some  reduction  compounds  of  tin, 
under  the  direction  of  Professor  A.  M.  Brumbach.  Following  a  sum- 
mer's work  in  the  final  inspection  department  of  the  Dayton  Engineer- 
ing Laboratories,  Dayton,  Ohio,  he  went  in  October,  1913,  to  the 
Iowa  State  College  of  Agriculture  and  Mechanic  Arts  as  an  instructor 
tor  in  the  department  of  chemistry.  He  remained  there  as  instructor 
in  home  economics  and  food  chemistry  until  September,  1917,  when 
he  returned  to  Denison  University  as  Assistant  Professor  of  chemis- 
try. He  had  taken  work  in  the  graduate  school  of  Chicago  Univer- 
sity during  the  summer  quarters  of  1914  and  1916. 

After  a  three  months'  training  at  Fort  Sheridan,  Illinois,  during  the 
summer  of  1918  he  was  given  a  certificate  of  Commission  as  second 
lieutenant  and  sent  back  to  teach  chemistry  at  Denison. 

He  enrolled  as  a  student  in  the  graduate  school.  University  of  Min- 
nesota, in  September,  1921.  Having  been  granted  a  fellowship  by  the 
American  Institute  of  Baking,  Minneapolis,  the  work  on  his  thesis  was 
carried  out  in  their  laboratories,  under  the  direction  of  Dr.  R.  A.  Gort- 
ner  and  Dr.  C.  M.  Bailey  of  the  University  of  Minnesota,  Division  of 
Agricultural  Biochemistry. 

In  May,  1922  he  presented  to  the  faculty  of  the  graduate  school  a 
thesis  on  "The  diastatic  enzymes  o^  wheat  flour  and  their  relation  to 
flour  strength,"  in  partial  fulfillment  of  the  requirements  for  the  degree 
of  Doctor  of  Philosophy.  The  degree  was  conferred  June  14th,  1922 
by  the  University  of  Minnesota. 


85 


ACKNOWLEDGMENT. 

The  author  wishes  to  express  his  gratitude  and  appreciation  for  the 
inspiration  and  help  of  Dr.  Ross  Aiken  Gortner,  under  whose  direction 
this  investigation  was  carried  out.  Grateful  acknowledgment  is  like- 
wise made  for  the  assistance  and  counsel  of  Dr.  Clyde  H.  Bailey,  and 
to  the  American  Institute  of  Baking  for  the  grant  of  a  fellowship  and 
for  the  facilities  made  available  for  the  prosecution  of  this  work. 

L.  A.  RUMSEY. 


86 


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